JP2024504240A - Tightly controlled inducible expression system for biopharmaceutical production using stable cell lines - Google Patents

Abstract

The present disclosure relates to tightly controlled, inducible expression systems useful for the inducible production of one or more RNAs or proteins of interest, including the production of biologics such as recombinant proteins, vaccines or viral vectors. Also provided are cell lines and kits useful for producing said RNA or protein, as well as methods for producing said cell lines and methods for inducing production of said RNA or protein. [Selection diagram] Figure 1

Description

[0003] The present disclosure relates to gene expression systems for inducible expression of one or more RNAs or proteins of interest in mammalian cells. Also disclosed are cell lines and methods for the inducible production of biologics such as recombinant proteins, vaccines, or viral vectors in mammalian cells.

[0004] Various biological products, such as recombinant proteins, vaccines, and viral vectors, are often produced for clinical applications using cultured mammalian cells. The availability of cell lines capable of efficiently producing such biological products without the need for transfection or infection greatly facilitates the manufacturing process. However, because some of the components that make up these biological preparations are cytotoxic, it is often difficult to generate such cell lines. As a result, the constitutive synthesis of these components prevents proper cell growth. Such cells are unstable or produce low amounts of product. Therefore, it is not suitable for manufacturing.

[0005] In order to produce cytotoxic biological products using stable cell lines, the tetracycline gene switch (Gossen and Bujard, 1992), the cumate gene switch (Mullick et al., 2006), etc. Alternatively, it is necessary to control transcription of the gene using an inducible expression system such as the coumermycin gene switch (Zhao et al., 2003). In such an inducible expression system, transcription of the gene encoding the biological product is inactive (the expression system is turned off) during cell isolation and propagation. When synthesis of a biological product is required, transcription is activated (the expression system is turned on) by adding an inducer (eg doxycycline or cumate). To use an inducible expression system, the cell must integrate into the chromosome the gene(s) encoding the regulatory elements of the gene switch, such as a transactivator or a repressor.

[0006] Several inducible expression systems have been developed (eg, coumate, tetracycline and coumermycin gene switches). The main drawbacks of some inducible expression systems are their leaky nature (the gene of interest is transcribed at low levels even without induction) and/or their mild efficacy (if induced, the expression system gives only weak gene expression).

[0007] The present disclosure combines the coumermycin gene switch (Zhao et al., 2003) and the coumate gene switch (Mullick et al., 2006) to provide dual regulation of target gene expression and reduced leakiness. thereby providing an expression system for inducible expression of one or more RNAs or proteins of interest. We demonstrated this dual-switch expression system using the CymR gene controlled by a constitutive promoter and the λR-GyrB gene under the control of a cumate-inducible promoter. Therefore, transcription of λR-GyrB was controlled by the cumate gene switch. In the absence of cumate, the CymR repressor bound to the cumate-inducible promoter CMV5CuO and inhibited transcription of λR-GyrB. Transcription of the λR-GyrB gene (and thus production of the λR-GyrB transactivator) was induced by the addition of cumate, and CymR was released from the CMV5CuO promoter. In this gene expression system, transcription of the gene(s) encoding the biological product(s) produced is driven by the coumermycin inducible promoter 12xlambda-CMVmin (or 12xlambda-TPL, or variants of these promoters). controlled by. This promoter was activated by binding of the λR-GyrB dimer. λR-GyrB formed a dimer in the presence of coumermycin. In the absence of coumermycin, λR-GyrB remained monomeric and did not bind to 12xlambda-CMVmin and thus did not activate transcription from 12xlambda-CMVmin. In summary, in the cumate/coumermycin dual gene switch, induction involves two inducers: cumate, which releases inhibition by CymR and allows synthesis of λR-GyrB, and dimerization of λR-GyrB and 12xlambda. - achieved by adding coumermycin, which allows transcription from CMVmin.

[0008] One aspect of the present disclosure includes an inducible expression system comprising: a first expression comprising a nucleic acid molecule encoding a coumat repressor protein operably linked to a constitutive promoter and a polyadenylation signal. a second expression cassette comprising a nucleic acid molecule encoding a coumermycin chimeric transactivator protein operably linked to a coumermycin inducible promoter and a polyadenylation signal; and (i) a coumermycin inducible promoter, a cloning site, and a polyadenylation signal, wherein the cloning site is for insertion of a nucleic acid molecule encoding a first RNA or protein of interest operably linked to the coumermycin-inducible promoter and the polyadenylation signal. or (ii) a nucleic acid molecule encoding a first RNA or protein of interest operably linked to a coumermycin-inducible promoter and a polyadenylation signal.

[0009] In one embodiment, the constitutive promoter is the human ubiquitin C (UBC) promoter, the human elongation factor 1α (EF1A) promoter, the human phosphoglycerate kinase 1 (PGK) promoter, the simian virus 40 early promoter (SV40), selected from the group consisting of the β-actin promoter, the cytomegalovirus immediate early promoter (CMV), the hybrid CMV enhancer/β-actin promoter (CAG), and variants thereof.

[0010] In one embodiment, the cumate repressor protein is a nucleic acid molecule comprising the amino acid sequence set forth in SEQ ID NO: 2, or a functional variant thereof, or comprising the nucleotide sequence set forth in SEQ ID NO: 1, or a functional variant thereof. coded by

[0011] In one embodiment, the cumate-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO: 5, or a functional variant thereof.

[0012] In one embodiment, the coumermycin chimeric transactivator protein comprises the amino acid sequence set forth in SEQ ID NO: 14, or a functional variant thereof, or comprises the nucleotide sequence set forth in SEQ ID NO: 13, or a functional variant thereof. encoded by a nucleic acid molecule that contains

[0013] In one embodiment, the coumermycin-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO: 9, or a functional variant thereof, or comprises the nucleotide sequence set forth in SEQ ID NO: 10, or a functional variant thereof.

[0014] In one embodiment, the coumermycin-inducible promoter further comprises a tripartite leader (TPL) and/or major late promoter (MLP) enhancer. In one embodiment, the coumermycin-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO: 11 or a functional variant thereof.

[0015] In one embodiment, the coumermycin-inducible promoter further comprises a human β-globin intron. In one embodiment, the coumermycin-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO: 12 or a functional variant thereof.

[0016] In one embodiment, the third expression cassette includes a nucleic acid molecule encoding the first RNA or protein of interest operably linked to a coumermycin-inducible promoter and a polyadenylation signal. In one embodiment, the third expression cassette encodes a recombinant protein.

[0017] In one embodiment, the expression system further comprises a fourth expression cassette comprising a nucleic acid molecule encoding a second RNA or protein of interest operably linked to a promoter and a polyadenylation signal.

[0018] In one embodiment, the expression system further comprises a fifth expression cassette comprising a third RNA or a nucleic acid molecule encoding a protein of interest operably linked to a promoter and a polyadenylation signal.

[0019] In one embodiment, the promoter of the fourth and/or fifth expression cassette is a coumermycin inducible promoter.

[0020] In one embodiment, the promoter of the fourth and/or fifth expression cassette is a constitutive promoter.

[0021] In one embodiment, the expression system encodes one or more components of a viral vector.

[0022] In one embodiment, the third expression cassette encodes a lentiviral REV protein, the promoter of the fourth expression cassette is a coumermycin inducible promoter, the fourth expression cassette encodes a viral envelope protein, The fifth expression cassette encodes lentiviral Gag/pol. In one embodiment, the viral envelope protein is VSVg, optionally VSVg-Q96H-I57L.

[0023] In one embodiment, the third expression cassette encodes a viral envelope protein, the promoter of the fourth expression cassette is a coumermycin inducible promoter, and the fourth expression cassette encodes a lentiviral Gag/pol. , the fifth expression cassette encodes the lentiviral REV protein. In one embodiment, the viral envelope protein is VSVg, optionally VSVg-Q96H-I57L.

[0024] In one embodiment, the third expression cassette encodes Rep40 or Rep52, the fourth expression cassette encodes Rep68 or Rep78, and the fourth expression cassette is under the control of a coumermycin inducible promoter.

[0025] In one embodiment, the third expression cassette encodes Rep52, the fourth expression cassette encodes Rep68, the fifth expression cassette encodes Rep78, and the fourth and fifth expression cassettes It is under the control of a coumermycin inducible promoter.

[0026] In one embodiment, the third expression cassette encodes an antibody heavy chain or a portion thereof and the fourth expression cassette encodes an antibody light chain or a portion thereof.

[0027] Another aspect of the disclosure includes a method of producing a mammalian cell for producing RNA or protein of interest. In one embodiment, the method comprises the step of introducing an expression system and a selectable marker described herein into a mammalian cell, by applying selective pressure on the cell to select for cells carrying the selectable marker. , including the steps of selecting cells harboring the expression system and creating mammalian cells for production of the RNA or protein of interest.

[0028] In one embodiment, the method includes the steps of isolating individual cells carrying the selectable marker and expression system; and culturing the individual cells to form a population of cells carrying the selectable marker and expression system. Further steps include creating.

[0029] In one embodiment, the method comprises: a) introducing into a mammalian cell a first expression cassette and a first selectable marker of an expression system described herein; b) c) selecting cells carrying the first expression cassette by applying selective pressure on the cells to select cells carrying the first selectable marker; c) selecting cells carrying the first expression cassette; isolating the individual population; d) culturing the first cell to obtain a first cell population comprising the first expression cassette; e) injecting the cells of the first cell population with introducing a second expression cassette of the described expression system and a second selectable marker; f) applying selective pressure to the cells to select cells carrying the second selectable marker; g) isolating a second individual cell containing the second expression cassette; h) culturing the second individual cell to express the second expression cassette; g) isolating a second individual cell containing the second expression cassette; obtaining a second cell population comprising the cassette; i) introducing into cells of the second cell population a third expression cassette and a third selectable marker of the expression system described herein; j ) selecting cells carrying the third expression cassette by applying selective pressure on the cells to select cells having the third selectable marker; k) selecting cells carrying the third expression cassette; isolating individual cells; l) culturing a third individual cell to obtain a third population of cells containing a third expression cassette; Steps to generate animal cells.

[0030] In one embodiment, the fourth expression cassette, and optionally the fifth expression cassette, of the expression system described herein is introduced into the cell after step i) or step l).

[0031] In one embodiment, a method of producing a mammalian cell for producing an RNA or protein of interest comprises: a) injecting a first molecule of an expression system described herein into a mammalian cell. introducing an expression cassette, a second expression cassette of the expression system described herein, and a first selectable marker; b) selecting cells to select cells carrying the first selectable marker; applying pressure, thereby selecting cells carrying the first and second expression cassettes; c) isolating the first individual cells containing the first expression cassette and the second expression cassette; d) culturing individual cells to obtain a first cell population comprising a first expression cassette and a second expression cassette; e) injecting the cells of the first cell population with expression as described herein. introducing a third expression cassette and a second selectable marker of the system; f) applying selective pressure to the cells to select cells carrying the second selectable marker, thereby inhibiting the expression of the third g) isolating a second individual cell containing the third expression cassette; h) culturing the second individual cell containing the third expression cassette. Generating mammalian cells for production of RNA or protein of interest by obtaining a second cell population.

[0032] In one embodiment, the fourth expression cassette of the expression system described herein, and optionally the fifth expression cassette of the expression system described herein, is added in step e) or step h). after which it is introduced into the cells.

[0033] In one embodiment, the expression system or one or more expression cassettes of an expression system described herein is produced by transfection, transduction, infection, electroporation, sonoporation, nucleofection, or introduced into cells by microinjection.

[0034] Other embodiments include cells comprising the expression systems described herein or cells produced by the methods described herein.

[0035] In one embodiment, the cells are human cells, optionally human embryonic kidney (HEK)-293 cells or derivatives thereof, Chinese hamster ovary (CHO) cells or derivatives thereof, VERO cells or derivatives thereof, HeLa cells. or a derivative thereof, A549 cells or a derivative thereof, a stem cell or a derivative thereof, or a neuron or a derivative thereof.

[0036] Other embodiments include methods of producing RNA or proteins of interest. In one embodiment, the method comprises culturing a cell comprising an expression system described herein in the presence of a cumate effector molecule and a coumermycin effector molecule, wherein the expression system of the cell is A third expression cassette encodes the RNA or protein of interest, and the RNA or protein of interest is produced.

[0037] In one embodiment, the cumate effector molecule is cumate, and optionally the cumate is present at a concentration of about 1 to about 200 μg/ml, about 50 to about 150 μg/ml, or about 100 μg/ml.

[0038] In one embodiment, the coumermycin effector molecule is coumermycin, and optionally the coumermycin is present at a concentration of about 1 to about 30 nM, about 5 to about 20 nM, or about 10 nM.

[0039] In one embodiment, the cells are grown in suspension and/or in the absence of serum.

[0040] One embodiment includes a viral packaging cell comprising an expression system described herein. In one embodiment, the viral packaging cell is a lentiviral packaging cell. In other embodiments, the viral packaging cell is an adeno-associated virus (AAV) packaging cell.

[0041] In one embodiment, the viral packaging cell further comprises a viral construct carrying a gene of interest.

[0042] Other embodiments include methods of producing viral vectors that include the steps of: introducing into a viral packaging cell described herein a viral construct having a gene of interest; and a cumate effector molecule. and producing a viral vector by culturing the cells in the presence of a coumermycin effector molecule.

[0043] In one embodiment, the cumate effector molecule is cumate and/or the coumermycin effector molecule is coumermycin.

[0044] In one embodiment, the virus packaging cells are grown in suspension and/or in the absence of serum.

[0045] In one embodiment, the selectable marker is introduced into the viral packaging cell along with the viral construct, and the method applies selective pressure to select cells that carry the selectable marker, thereby displacing the viral construct. The method further comprises selecting cells harboring the viral construct, and optionally isolating individual cells containing the viral construct and culturing the individual cells containing the viral construct to obtain a population of cells containing the viral construct.

[0046] In one embodiment, the viral packaging cell is a lentiviral packaging cell and the viral construct is a lentiviral construct.

[0047] In one embodiment, the viral packaging cell is an AAV packaging cell and the viral construct is an AAV construct.

[0048] Additional embodiments include methods of creating expression capable mammalian cell lines. In one embodiment, the method comprises: administering to a mammalian cell a first expression cassette of an expression system described herein, a second expression cassette of an expression system described herein, and a first expression cassette of an expression system described herein; selecting cells carrying the first and second expression cassettes by applying selective pressure on the cells to select cells carrying the first selectable marker; isolating individual cells containing the first and second expression cassettes; and culturing the individual cells to produce a mammalian cell line containing the first and second expression cassettes. Steps to create strains.

[0049] In one embodiment, a method of creating an expressible mammalian cell line comprises: injecting into a mammalian cell a first expression cassette of an expression system described herein and a first selectable introducing a marker; selecting cells carrying the first expression cassette by applying selective pressure on the cells to select cells carrying the first selectable marker; isolating a first individual cell comprising the first individual cell; culturing the first individual cell to obtain a first cell population comprising the first expression cassette; introducing a second expression cassette of the expression system described herein and a second selectable marker; applying selective pressure to the cells to select cells carrying the second selectable marker; and culturing the second individual cells to obtain a second population of cells comprising the second expression cassette. Obtaining animal cell lines.

[0050] Other embodiments include mammalian cells comprising a first expression cassette of an expression system described herein and a second expression cassette of an expression system described herein.

[0051] In one embodiment, the cells are human cells, optionally human embryonic kidney (HEK)-293 cells or derivatives thereof.

[0052] A further embodiment includes a method of producing an RNA or protein of interest, the method comprising: a first expression cassette of an expression system described herein and an expression system described herein. introducing a second expression cassette of the system and a cell comprising a third expression cassette of the expression system described herein and a selectable marker; selecting cells harboring the first, second and third expression cassettes of the expression system by applying selective pressure to the individual cells containing the first, second and third expression cassettes; isolating the cells and culturing the individual cells to generate a population of cells comprising the first, second and third expression cassettes; and in the presence of a cumate effector molecule and a coumermycin effector molecule; Cultivating cells containing the first, second, and third expression cassettes, in which RNA or protein of interest is produced. In one embodiment, the cumate effector molecule is cumate and/or the coumermycin effector molecule is coumermycin. In one embodiment, the cells are grown in suspension and/or in the absence of serum.

[0053] Still other embodiments of the disclosure include kits containing the expression systems described herein. In one embodiment, the kit includes: a first plasmid comprising a first expression cassette of an expression system described herein; a second plasmid comprising a second expression cassette of an expression system described herein; and a third plasmid comprising a third expression cassette of the expression system described herein. In one embodiment, the kit comprises a cell comprising a first expression cassette and a second expression cassette of an expression system described herein, and a third expression cassette of an expression system described herein. Contains plasmids containing.

[0054] In one embodiment, the third expression cassette includes a coumermycin-inducible promoter, a cloning site, and a polyadenylation signal, wherein the cloning site is operably linked to the coumermycin-inducible promoter and the polyadenylation signal. for inserting a nucleic acid molecule encoding a first RNA or protein of interest. In one embodiment, the third expression cassette comprises a nucleic acid molecule encoding the first RNA or protein of interest operably linked to a coumermycin inducible promoter and a polyadenylation signal.

[0055] In one embodiment, a cell comprising a first expression cassette and a second expression cassette of an expression system described herein is a cell comprising a third, fourth and second expression cassette of an expression system described herein. and/or a fifth expression cassette, wherein the third, fourth and/or fifth expression cassette encodes an RNA or protein of interest.

[0056] In one embodiment, the kit includes a viral packaging cell comprising an expression system described herein, and a viral construct.

[0057] In one embodiment, the kit further comprises a cumate effector molecule, optionally cumate, and/or a coumermycin effector molecule, optionally coumermycin.

[0058] The preceding sections are provided by way of example only and are not intended to limit the scope of this disclosure or the appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be appreciated by those skilled in the art in light of the claims, description, and examples. For example, the various aspects and embodiments of this disclosure may be utilized in numerous combinations, all of which are expressly contemplated by this specification. These additional benefit objects and embodiments are expressly included within the scope of this disclosure. Publications and other materials used herein to provide background to the disclosure and, in certain cases, to provide additional details regarding the practice, are incorporated by reference. For convenience, they are listed in the attached references section.

[0059] Further objects, features, and advantages of the present disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying figures that illustrate exemplary embodiments of the present disclosure:
FIG. 1 is a schematic diagram of the gene regulation mechanism of an embodiment of the coumate/coumermycin gene switch. A) 293SF-CymR/λR-GyrB cells were engineered to constitutively produce the repressor of the cumate gene switch (CymR). This cell also contains the gene for the coumermycin chimeric transactivator (λR-GyrB) under the control of the CMV5CuO promoter. In the absence of cumate, CymR binds to the CMV5CuO promoter and inhibits transcription of λR-GyrB. Addition of cumate releases CymR from the promoter and λR-GyrB is transcribed. B) In the presence of coumermycin, λR-GyrB forms a dimer and binds to the 12xlambda operator (12xλOp), activating transcription of the transgene of interest. Transcription can be prevented by adding novobiocin to cells to dissociate the λR-GyrB dimer. Figure 2 shows a diagram of the constructs used to generate 293SF-CymR, 293SF-CymR/λR-GyrB and 293SF-CymR/rcTA cells. A) CymR constructs used to generate 293SF-CymR, 293SF-CymR/λR-GyrB, and 293SF-CymR/rcTA cells. The CymR repressor coding sequence is controlled by a strong constitutive promoter (CMV5). B) λR-GyrB construct used to generate 293SF-CymR/λR-GyrB cells. The coding sequence of λR-GyrB is controlled by the CMV5CuO promoter. C) rcTA construct used to generate 293SF-CymR/rcTA cells. pA: polyadenylation signal. Figure 3 shows a diagram of the LV-encoding transfer vector used in this study. A) LV-CMV5CuO-GFP transfer vector. B) Transfer vector for LV-12xlambda-TPL-GFP. C) Transfer vector for LV-CR5-GFP. D) Transfer vector for LV-CMV-GFP. Note: The transfer vectors shown in A), B) and C) produce conditional self-inactivating lentivirus (cSIN). 5'LTR and 3'LTR: long terminal repeat located at the 5' or 3' end of lentivirus, respectively; CMV: CMV promoter; R: R region of LTR; U5: U5 region of LTR: Tet: tetracycline promoter; ψ : encapsidation signal; RRE: Rev response element; cPPT: central polypurine track; GFP: green fluorescent protein; WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element; SD and SA: splice donor and acceptor, respectively; CMV5CuO and CR5 cumate control Promoter; 12xλ-TPL: 12xLambda-TPL coumermycimin-regulated promoter Figure 4 shows a schematic diagram of the mechanism of gene regulation in 293SF-CymR/rcTA cells. A) 293SF-CymR/rcTA cells were engineered to constitutively produce the repressor of the cumate gene switch (CymR). The cells also contain the gene for reverse transactivator (rcTA) under the control of the CMV5CuO promoter. In the absence of cumate, CymR binds to the CMV5CuO promoter and inhibits transcription of the rcTA gene. Addition of cumate releases CymR from the promoter, allowing transcription of rcTA. B) In the presence of cumate, rcTA binds to the CR5 promoter and activates transcription of the transgene of interest (reporter). Figure 5 shows that the dual coumermycin/coumate gene switch provides better induction levels compared to the coumate gene switch. A) 293SF-CymR clones (198-2, 198-10, 169-4, 169-C1, CA7), B) 293SF-CymR/rcTA clones (G3 and G11) and C) 293SF-CymR/λR- GyrB clones (7-2, 7-3, 7-10) were transduced with LV-CMV5CuO-GFP, LV-CR5-GFP and LV-12xlambda-TPL-GFP, respectively. After transduction, GFP expression levels were analyzed by flow cytometry. The fluorescence index of the cell population in the absence (Off) and presence (On) of the inducing agent was compared. The On/Off ratio for each clone is shown as numbers above the bar (B,C) or by a line (A). The On/Off ratios of the 293SF-CymR, 293SF-CymR/rcTA, and 29SF-CymR/λR-GyrB clones varied between 15-30, 60-100, and 2621-3877, respectively. 293SF are 293SF cells transduced with LV (no switch); T0 and T2 are cells maintained in culture for 1 week and 8 weeks, respectively. Figure 5 shows that the dual coumermycin/coumate gene switch provides better induction levels compared to the coumate gene switch. A) 293SF-CymR clones (198-2, 198-10, 169-4, 169-C1, CA7), B) 293SF-CymR/rcTA clones (G3 and G11) and C) 293SF-CymR/λR- GyrB clones (7-2, 7-3, 7-10) were transduced with LV-CMV5CuO-GFP, LV-CR5-GFP and LV-12xlambda-TPL-GFP, respectively. After transduction, GFP expression levels were analyzed by flow cytometry. The fluorescence index of the cell population in the absence (Off) and presence (On) of the inducing agent was compared. The On/Off ratio for each clone is shown as numbers above the bar (B,C) or by a line (A). The On/Off ratios of the 293SF-CymR, 293SF-CymR/rcTA, and 29SF-CymR/λR-GyrB clones varied between 15-30, 60-100, and 2621-3877, respectively. 293SF are 293SF cells transduced with LV (no switch); T0 and T2 are cells maintained in culture for 1 week and 8 weeks, respectively. Figure 5 shows that the dual coumermycin/coumate gene switch provides better induction levels compared to the coumate gene switch. A) 293SF-CymR clones (198-2, 198-10, 169-4, 169-C1, CA7), B) 293SF-CymR/rcTA clones (G3 and G11) and C) 293SF-CymR/λR- GyrB clones (7-2, 7-3, 7-10) were transduced with LV-CMV5CuO-GFP, LV-CR5-GFP and LV-12xlambda-TPL-GFP, respectively. After transduction, GFP expression levels were analyzed by flow cytometry. The fluorescence index of the cell population in the absence (Off) and presence (On) of the inducing agent was compared. The On/Off ratio for each clone is shown as numbers above the bar (B,C) or by a line (A). The On/Off ratios of the 293SF-CymR, 293SF-CymR/rcTA, and 29SF-CymR/λR-GyrB clones varied between 15-30, 60-100, and 2621-3877, respectively. 293SF are 293SF cells transduced with LV (no switch); T0 and T2 are cells maintained in culture for 1 week and 8 weeks, respectively. Figure 6 shows a diagram of the expression cassette used to construct the LV packaging cell line. To construct the LV packaging cell line, the following components were integrated into the chromosome of 293SF-CymR/λR-GyrB: i) the HIV Rev gene under the control of the coumermycin-inducible promoter 13xlambda-TPL; ii) the HIV Gag/pol gene controlled by the constitutive hybrid CMV enhancer/β-actin promoter (CAG) or the 11xlambda-hbgmin promoter; and iii) the vesicular stomatitis virus glycoprotein gene (VSVg) controlled by the 13xlambda-TPL. Addition of coumate and coumermycin induces transcription of Rev, VSVg and Gag/pol (when controlled by 11xlambda-hgbmin). Note: The presence of Rev is required for nuclear export of intact Gag/pol RNA. Therefore, efficient synthesis of Gag/pol polypeptides is dependent on the expression of Rev. Figure 7 shows lentivirus production after transfection of packaging cells. Clones of packaging cells grown in suspension culture using serum-free medium were transfected with the transfer vector (plasmid) for LV-CMV-GFP (FIG. 3D). Cells were induced with coumate and coumermycin, and culture medium was harvested 3 days after transfection. After transduction of HEK293 cells, LVs in the culture medium were quantified by flow cytometry. Bars are infectious titers (transducing units [TU] per mL) in culture medium. Transfection efficiency (% of GFP positive cells) is shown in A) with light white squares. A) Clone of packaging cells created using a plasmid encoding 11xlambda-hbgmin-Gag/pol. B) Clone of packaging cells created using a plasmid encoding CAG-Gag/pol. Note: Both packaging cell lines (A and B) yielded clones capable of producing LVs at titers of 1.0 x 10 ⁷ TU/ml or higher. Figure 7 shows lentivirus production after transfection of packaging cells. Clones of packaging cells grown in suspension culture using serum-free medium were transfected with the transfer vector (plasmid) for LV-CMV-GFP (FIG. 3D). Cells were induced with coumate and coumermycin, and culture medium was harvested 3 days after transfection. After transduction of HEK293 cells, LVs in the culture medium were quantified by flow cytometry. Bars are infectious titers (transducing units [TU] per mL) in culture medium. Transfection efficiency (% of GFP positive cells) is shown in A) with light white squares. A) Clone of packaging cells created using a plasmid encoding 11xlambda-hbgmin-Gag/pol. B) Clone of packaging cells created using a plasmid encoding CAG-Gag/pol. Note: Both packaging cell lines (A and B) yielded clones capable of producing LV at titers of 1.0 x 10 ⁷ TU/ml or higher. Figure 8 shows lentivirus production by production clones derived from packaging cell lines. An LV producing clone was created by transfecting a packaging cell (clone 3D4, FIG. 7B) with an transfer vector (plasmid) encoding LV-CMV-GFP (FIG. 3D). The resulting LV (LV-CMV-GFP) expresses GFP under the control of the constitutive CMV promoter. Clones that stably integrated the plasmid were isolated and grown in suspension culture using serum-free medium. Production of LV-CMV-GFP was induced by addition of cumate and coumermycin to the culture medium. The titer of LV-CMV-GFP in the culture medium was measured 3 days after introduction into HEK293 cells, and GFP-positive cells were quantified by flow cytometry. Titers were expressed as transduction units (TU) per ml of non-enriched culture medium. Note: Some clones (1E9, 3E9, 2G11, 1F3, 2C8 and 1E8) produce LV-CMV-GFP at titers of 1.0×10 ⁸ TU/ml of culture medium or higher. Figure 9 shows regulation of LV gene expression in packaging cells. Western blot analysis of the expression of VSVg (A), Rev (B) and Gag (C) by packaging cells (clone 3D4, Figure 7) after induction with coumate and coumermycin. The same amount of cell lysate was used for each sample. Cells were harvested before induction (0) and at 24, 48, and 72 hours after induction in the absence or presence of sodium butyrate (added 18 hours after induction). 293SF-CymR/λR-GyrB cells were used as a negative control (CT). The positions of VSVg, Rev, Gag polyprotein (GAG PP), and p24 are indicated by arrows. MW: Molecular weight marker in kDa. Note the presence of a non-specific band (*) in the negative control when using the VSVg antibody. Figure 10 shows a schematic diagram of the components used to produce AAV. A) Constructs expressing Rep52, Rep68, and Rep78 used to generate 293SF-Rep cells. Each Rep gene is controlled by the 13xlambda-TPL promoter (13xλ-TPL). B) Plasmid used to produce AAV by transient transfection of 293SF-Rep. pCMV-CAP encodes the AAV CAP gene controlled by the CMV promoter. pAAV-GFP is an expression vector that produces GFP controlled by CMV. pHelper carries adenovirus helper genes. Figure 11 shows a Western blot of REP protein expression by the 293SF-Rep clone. Cell lysates of stably transfected 293SF-Rep clones (clones 13, 18, 35 and 36) were analyzed by Western blotting using antibodies against REP. Expression was induced using different concentrations of cumate and coumermycin. Uninduced cells served as a negative control. The positions of Reps 78, 68, and 52 are indicated by arrows. Note: No significant expression of REP is observed without induction. MW: molecular weight marker. Figure 12 shows optimization of AAV production after transient transfection of 293SF-Rep cells (clone 13). 293SF-Rep cells grown in suspension culture in serum-free medium were transfected with different amounts (expressed in μg/ml) of pCMV-CAP (pVR46-CAP), pHelper and pAAV-GFP (see Figure 10). ). After transfection, cells were induced with coumate and coumermycin for 3 days. Cell lysates containing the produced AAV were used to transduce HEK293 cells and then analyzed by flow cytometry. Data were expressed as infectious virus particles (IVP) per ml of cell culture. The best conditions were #5 and #16, which gave a titer of 2.5 x 10 ⁷ IVP/ml. Figure 13 shows the sequence (upper strand) and complementary sequence (lower strand) of promoter 12xLambda-CMVmin (SEQ ID NO: 9). The positions of the 12-copy lambda operator (12xlambdaOP) and the CMV minimal promoter are shown in the nucleotide sequence of 12xLambda-CMVmin. Figure 14 shows the sequence (upper strand) and complementary sequence (lower strand) of promoter 13xLambda-CMVmin (SEQ ID NO: 10). The positions of the 13-copy lambda operator (13xlambdaOP) and the CMV minimal promoter are shown in the nucleotide sequence of 13xLambda-CMVmin. Figure 15 shows the sequence (upper strand) and complementary sequence (lower strand) of promoter 13xLambda-TPL (SEQ ID NO: 11). The positions within the small intron of the 13-copy lambda operator (13xlambdaOP), the CMV minimal promoter, the adenovirus tripartite leader (TPL) and the enhancer of the adenovirus major late promoter (MLP enhancer) are shown. Figure 16 shows the sequence (upper strand) and complementary sequence (lower strand) of promoter 11xlambda-hbgmin (SEQ ID NO: 12). 11 copies of the lambda operator (11xlambdaOP), the CMV minimal promoter, the adenovirus tripartite leader (TPL), the enhancer of the adenovirus major late promoter (MLP enhancer), the human β-globin intron (hBglobin-delta intron), and the chimeric intron. Locations of splice site donors and acceptors are shown. Figure 16 shows the sequence (upper strand) and complementary sequence (lower strand) of promoter 11xlambda-hbgmin (SEQ ID NO: 12). 11 copies of the lambda operator (11xlambdaOP), the CMV minimal promoter, the adenovirus tripartite leader (TPL), the enhancer of the adenovirus major late promoter (MLP enhancer), the human β-globin intron (hBglobin-delta intron), and the chimeric intron. Locations of splice site donors and acceptors are shown.

[Description of various embodiments]
[0076] The following is a detailed description provided to assist those skilled in the art in implementing the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the disclosure. All publications, patent applications, patents, figures, and other references mentioned herein are expressly incorporated by reference in their entirety.

I. definition
[0077] As used herein, the following terms may have the meanings ascribed to them unless specified otherwise. However, it is to be understood that other meanings known or understood by those skilled in the art are also possible and within the scope of this disclosure. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0078] When a range of values is provided, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range up to one-tenth of a unit of the lower limit, and Other stated or intervening values within the stated range are understood to be included within this specification. A range from any lower limit to any upper limit is envisioned.

[0079] As used herein, the term "about" is used to account for experimental error and variations that would be expected by those skilled in the art. For example, "about" can mean plus or minus 10%, or plus or minus 5% of the referenced indicated value.

[0080] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0081] As used herein, the phrase "and/or" refers to "either or both" of the elements so conjoined, i.e., present conjointly in some instances and in other instances. should be understood to mean elements that exist separately. Multiple elements listed with "and/or", ie, "one or more" of the elements so conjoined, should be construed similarly. Other elements may optionally be present other than those specifically identified by the "and/or" phrase, whether or not related to the specifically identified elements.

[0082] As used herein and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" is inclusive, i.e. includes a number or at least one of the elements in the list, but more than one. and, optionally, shall be construed to include items not further listed. A unitary term clearly indicating the opposite, such as "only one of", "exactly one of", or "consisting of" when used in a claim, Refers to containing exactly one element out of a number or list of elements. Generally, as used herein, the term "or" includes "any," "one of," "only one of," or "exactly one of," etc. shall be construed as indicating an exclusive alternative (i.e., "one or the other, but not both") only when preceded by an exclusivity term.

[0083] As used herein, "comprising", "including", "carrying", "having", "containing", "accompaniing" All transitional phrases such as "including", "holding", "composed of", etc. are understood to be open-ended, ie, to mean including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0084] As used herein, the phrase "at least one" with respect to a list of one or more elements means at least one selected from any one or more elements in the list of elements. is to be understood as meaning an element, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not necessarily include any of the elements in the list of elements. A combination of these is not excluded. This definition also applies to elements other than those specifically identified in the list of elements referred to by the phrase "at least one," regardless of whether they are related to the specifically identified element. It allows for it to exist by choice.

[0085] Also, in certain methods described herein that include more than one step or act, unless otherwise indicated, the order of the method steps or acts is the order in which the method steps or acts are described. It should be understood that the order in which they appear is not necessarily limited.

II. expression system
[0086] The use of a dual coumermycin/coumate gene switch was found to provide tightly controlled expression of the protein of interest (higher On/Off ratio) compared to the use of the coumate switch alone. It was done. Accordingly, provided herein is an expression system containing a dual coumermycin/coumate gene switch that is useful for tightly controlled, inducible expression of RNA or proteins of interest. Accordingly, provided herein are a) a first expression cassette comprising a nucleic acid molecule encoding a cumate repressor protein operably linked to a constitutive promoter and a polyadenylation signal; b) cumate induction a second expression cassette comprising a nucleic acid molecule encoding a coumermycin chimeric transactivator protein operably linked to a coumermycin inducible promoter and a polyadenylation signal; a coumermycin-inducible promoter, a cloning site, and a polyadenylation signal for inserting a nucleic acid molecule encoding a first RNA or protein of interest operably linked to a coumermycin-inducible promoter, a cloning site, and a polyadenylation signal; An expression system comprising a third expression cassette comprising a nucleic acid molecule encoding at least one RNA or protein of interest operably linked to a promoter and a polyadenylation signal.

[0087] The term "expression cassette" refers to an RNA or protein-encoding DNA molecule operably linked to a promoter and a polyadenylation signal, and certain portions of the expression cassette are RNA (antisense RNA, long non- (e.g., a coding RNA or the genome of a virus such as a lentivirus) and/or capable of being transcribed as a messenger RNA that is subsequently translated into protein by the cellular machinery. The term "expression cassette" refers to a nucleic acid molecule that includes a promoter, a polyadenylation signal, and a cloning site for inserting a nucleic acid molecule encoding an RNA or protein of interest operably linked to the promoter and the polyadenylation signal. Also used to refer to.

[0088] As used herein, the term "nucleic acid molecule" and derivatives thereof are intended to include unmodified DNA or RNA or modified DNA or RNA. For example, the nucleic acid molecules or polynucleotides of the present disclosure include single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and single-stranded DNA. From hybrid molecules comprising RNA which is a mixture of regions and double-stranded regions, DNA and RNA which can be single-stranded or more typically double-stranded, or a mixture of single-stranded regions and double-stranded regions. can be configured. Additionally, a nucleic acid molecule can be composed of triple-stranded regions containing RNA or DNA, or both RNA and DNA. Nucleic acid molecules of the present disclosure may also include one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. DNA and RNA can be modified in a variety of ways, and thus "nucleic acid molecule" includes chemically, enzymatically, and metabolically modified forms. The term "polynucleotide" shall have the corresponding meaning.

[0089] As used herein, the term "cloning site" refers to a site into which a nucleic acid molecule of interest may be inserted or to which a nucleic acid molecule of interest may be joined using recombinant DNA technology (cloning). Refers to the part of a nucleic acid molecule. In the case of an expression cassette, the cloning site may be located between the promoter and the polyadenylation signal such that the nucleic acid molecule of interest is operably linked to the promoter and the polyadenylation site so that it can be cloned into the expression cassette. . Several cloning techniques are known to those skilled in the art, and the cloning site has the necessary characteristics (restriction endonuclease site(s), recombinase recognition site(s)) to allow insertion of the desired nucleic acid molecule at the cloning site. or blunt or protruding end(s)). The cloning site may be, for example, a multiple cloning site (MCS) or a polylinker region containing multiple unique restriction enzyme recognition sites to allow insertion of the nucleic acid molecule of interest. Alternatively or additionally, the cloning site may include one or more cloning sites to enable DNA insertion by recombinant cloning using site-specific recombinase(s) such as integrase or Cre recombinase to catalyze the DNA insertion. One or more recombinase recognition sites can be included. Examples of recombinant cloning systems include Gateway® (Integrase), Creator® (Cre recombinase), and Echo Cloning® (Cre recombinase). It will be done. In some cloning strategies, the expression cassette or vector is provided as a linear molecule, and the blunt or overhanging ends of the nucleic acid molecule of interest are joined to the blunt or overhanging ends of the expression cassette or vector, e.g. by ligation or polymerase activity, to form a circular molecules can be formed. In this case, the blunt or overhanging ends of the expression cassette or vector can be considered together as a cloning site. Such an approach is commonly used for cloning PCR products.

[0090] As used herein, the term "operably linked" refers to a relationship between two components that enables the two components to function in their intended manner. For example, when DNA encoding an RNA of interest is operably linked to a promoter, the promoter drives expression of the encoded RNA.

[0091] The term "promoter" or "promoter sequence" generally refers to a regulatory DNA sequence that can be joined by an RNA polymerase to initiate transcription of a downstream (i.e., 3') sequence to produce RNA. Point. A suitable promoter may be derived from any organism and may be bound or recognized by any RNA polymerase. Suitable promoters are known to those skilled in the art. In some expression cassettes, the promoter is a constitutive promoter. Examples of constitutive promoters include human ubiquitin C (UBC), human elongation factor 1α (EF1A), human phosphoglycerate kinase 1 (PGK), vasoactive intestinal peptide (VIP), thymidine kinase (tk), heat shock proteins. (HSP), adenovirus major late promoter (MLP), mouse mammary tumor virus (MMTV), simian virus 40 early promoter (SV40), β-actin, cytomegalovirus immediate early promoter (CMV), hybrid CMV enhancer/β - actin promoter (CAG), or functional variants thereof. In some expression cassettes, the promoter is an inducible promoter and/or contains binding sequences for transactivators or repressors that activate or inhibit transcription, respectively. Examples of inducible promoters include coumate-inducible promoters and coumermycin-inducible promoters.

[0092] The term "cumate-inducible promoter" refers to a promoter that can be bound by a cumate repressor protein in the absence of a cumate effector molecule, such as CymR (SEQ ID NO: 1 and 2) or a functional variant thereof. . Binding of cumate effector molecules relieves transcriptional repression by cumate repressor proteins such as CymR. Kumate-inducible promoters are described, for example, in U.S. Pat. No. 7,745,592, and include minimal promoter sequences (e.g. TATA box and adjacent sequences) and at least one CymR operator sequence (CuO). CuO sequences include, for example, CuO P1 (SEQ ID NO: 4) or CuO P2 as described in US Pat. No. 7,745,592. In some embodiments, CuO with palindromic characteristics is used, such as CuO P2 (SEQ ID NO: 3). In some embodiments of the present disclosure, CMV5-CuO (SEQ ID NO: 5) or a functional variant thereof may be used.

[0093] As used herein, the term "coumate effector molecule" refers to a molecule that relieves transcriptional repression by cumate repressor proteins. Cumate effector molecules include cumate, p-ethylbenzoic acid, p-propylbenzoic acid, cumic acid, p-isobutylbenzoic acid, p-tert-butylbenzoic acid, p-N-dimethylaminobenzoic acid, and p- N-ethylaminobenzoic acid is mentioned. In one embodiment, the cumate effector molecule is cumate.

[0094] As used herein, the term "coumermycin-inducible promoter" refers to a promoter that can be bound by a coumermycin chimeric transactivator protein. Coumermycin-inducible promoters are described, for example, in U.S. Patent No. 8,377,900 and are derived from, for example, the CMV, VIP, SV40, tk, HSP, PGK, MLP, EF1a and MMTV promoters and functional variants thereof. Contains minimal promoter sequences (eg, a TATA box and flanking sequences) from a selected constitutive mammalian promoter, and at least one lambda operator (λOP) sequence (SEQ ID NO: 6). The coumermycin-inducible promoter may contain, for example, 1 to 13 copies of lambdaOp, optionally 11, 12 (SEQ ID NO: 7) or 13 (SEQ ID NO: 8) copies or more of lambdaOp. In one embodiment, the coumermycin inducible promoter is the minimal CMV promoter and several copies, such as 11, 12 or 13 copies of lambdaOP, such as 12xlambda-CMVmin or 13xlambda-CMVmin, or functional variants thereof. In other embodiments, the coumermycin-inducible promoter may include multiple copies of lambdaOP along with the minimal CMV promoter and adenoviral tripartite leader (TPL) and major late promoter (MLP) enhancers, e.g., SEQ ID NO: 11 and 15, or a functional variant thereof. In other embodiments, the coumermycin-inducible promoter includes 11 copies of lambdaOP, the minimal CMV promoter, the adenovirus TPL and MLP enhancers, and the human β-globin intron, for example, as described in SEQ ID NO: 12 and FIG. 16. The promoter may contain several copies of lambdaOp, or a functional variant thereof, associated with other downstream sequences, such as the promoter 11xlambda-hbgmin, including a portion.

[0095] As used herein, the term "coumermycin chimeric transactivator protein" refers to a protein that is capable of binding to a coumermycin inducible promoter in the presence of a coumermycin effector molecule. Binding of the coumermycin effector molecule results in dimerization of chimeric transactivator proteins such as λR-GyrB and transcriptional activation from the coumermycin-inducible promoter. Coumermycin chimeric transactivator proteins are described, for example, in U.S. Pat. No. 8,377,900, which fuse the N-terminal domain of the λ repressor (λR) with the DNA gyrase B subunit (GyrB); It can be constructed by subsequently fusing a transcriptional activation domain, also called a transactivation domain. The N-terminal domain of λR binds to lambdaOP as a dimer. The GyrB domain forms a dimer after binding with coumermycin, thus promoting the dimerization of the N-terminal domain of λR, which can then bind to lambdaOP and activate transcription via the activation domain. . For brevity, the coumermycin chimeric transactivator protein may be referred to herein simply as "λR-GyrB." Suitable transcriptional activation domains include those derived from transcription factors NFκB p65, VP16, B42 and Ga14. In one embodiment, the transactivation domain is from NFkB p65 as set forth in SEQ ID NO:15. In one embodiment, the coumermycin chimeric transactivator protein has a sequence as set forth in SEQ ID NO: 13 or 14, or a functional variant thereof. Suitable coumermycin effector molecules include coumermycin. To provide further suppression of protein expression or prevent leaky expression, dimerization and activation of the coumermycin chimeric transactivator protein can be inhibited by the addition of an inhibitor such as novobiocin.

[0096] As used herein, the term "polyadenylation signal" or "pA" generally refers to the polyadenylation signal (pA), which is the signal by which transcribed RNA is cleaved and a polyadenylation tail is added. This site has the effect of terminating the transcription of RNA such as messenger RNA (mRNA). Suitable pAs may be derived from any organism and are known to those skilled in the art. Examples of pA signals include rabbit beta globin pA (SEQ ID NO: 16), potent bovine growth hormone pA (BGHpA; SEQ ID NO: 17), and SV40 polyA signal (SEQ ID NO: 18).

[0097] As used herein, the term "functional variant" refers to a variant that performs substantially the same function in substantially the same way as a nucleic acid molecule or polypeptide disclosed herein. including modifications or chemical equivalents of nucleic acid sequences or proteins. For example, functional variants of the polypeptides disclosed herein include, but are not limited to, conservative amino acid substitutions.

[0098] As used herein, a "conservative amino acid substitution" is a replacement of one amino acid residue with another amino acid residue that has similar biochemical properties (e.g., charge, hydrophobicity, and size). It is something that will be done. Polypeptide variants also include additions and deletions to the polypeptide sequences disclosed herein. Additionally, variant nucleotide sequences include analogs and derivatives thereof.

[0099] In one embodiment, the present disclosure includes functional variants to the nucleic acid sequences disclosed herein. Functional variants include nucleotide sequences that hybridize to the above-described nucleic acid sequences under at least moderately stringent hybridization conditions.

[00100] "At least moderately stringent hybridization conditions" means that conditions are selected that promote selective hybridization between two complementary nucleic acid molecules in solution. The term "at least moderately stringent hybridization conditions" encompasses stringent hybridization conditions and moderately stringent hybridization conditions. Hybridization can occur to all or part of the nucleic acid sequence molecule. Hybridizing portions are typically at least 15 (eg, 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will understand that the stability of a nucleic acid duplex or hybrid is determined by the Tm, which in sodium-containing buffers is a function of sodium ion concentration and temperature (Tm = 81.5°C - 16.6(Log10[Na+])+0.41(%(G+C)-600/l), or similar formula). Therefore, the parameters of the washing conditions that determine the stability of the hybrid are sodium ion concentration and temperature. To identify molecules that are similar but not identical to known nucleic acid molecules, it can be assumed that a 1% mismatch reduces the Tm by approximately 1°C, e.g., nucleic acid molecules with greater than 95% identity. , the final wash temperature is reduced by about 5°C. Based on these considerations, one skilled in the art can readily select appropriate hybridization conditions. In some embodiments, stringent hybridization conditions are selected. As an example, the following conditions can be used to achieve stringent hybridization: 5x sodium chloride/sodium citrate (SSC)/5x Denhardt's solution/1.0% SDS with Tm based on the above formula. Hybridization is performed at -5°C followed by washing with 0.2xSSC/0.1% SDS at 60°C. Moderately stringent hybridization conditions include a wash step in 3xSSC at 42°C. However, it is understood that equivalent stringency can be achieved using other buffers, salts, and temperatures. Additional guidance on hybridization conditions can be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. , 2002, and Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[00101] In other embodiments, the functional variant nucleic acid sequence comprises a degenerate codon substitution or codon optimized nucleic acid sequence. As used herein, the term "degenerate codon substitution" refers to a variant nucleic acid sequence in which the second and/or third base of a codon is replaced with a different base that does not result in a change in the amino acid sequence encoded therein. Point. As used herein, the term "codon-optimized" refers to variant nucleic acid molecules that contain one or more degenerate codon substitutions that reflect the codon usage bias of a particular organism.

[00102] In other embodiments, the functional variant nucleic acid or protein sequence is at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least Includes sequences with 90%, or at least 95% sequence identity.

[00103] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment with a second amino acid or nucleic acid sequence). gaps may be introduced in the sequence of the first amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. Molecules are identical at a position in the first sequence if that position is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. Percent identity between two sequences is a function of the number of identical positions that the sequences share (ie, % identity = number of overlapping identical positions/total number of positions x 100%). In one embodiment, the two sequences are the same length. Determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm utilized to compare two sequences is described by Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U. S. A. 87:2264-2268, Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U. S. A. 90:5873-5877. Such an algorithm is described by Altschul et al. , 1990 in the NBLAST and XBLAST programs. BLAST nucleotide searches can be performed using, for example, the NBLAST nucleotide program parameters set to score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with XBLAST program parameters set to, for example, score-50, wordlength=3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gap alignment for comparison purposes, Altschul et al. , 1997, Nucleic Acids Res. 25:3389-3402, Gapped BLAST may be utilized. Alternatively, PSI-BLAST can be used to perform iterative searches that detect distant relationships between molecules. When utilizing the BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters for each program (eg, XBLAST and NBLAST) can be used (see, eg, the NCBI website). Another non-limiting example of a mathematical algorithm utilized for sequence comparisons is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing for gaps. Percent identity calculations typically only count exact matches.

[00104] The expression systems described herein can be used to express any RNA or protein of interest. Thus, in one embodiment, the expression system encodes the RNA or protein of interest. The RNA or protein of interest may be cytotoxic or result in decreased cell proliferation and/or viability. In one embodiment, the protein of interest is a recombinant protein.

[00105] The expression systems described herein can be used to express more than one RNA or protein of interest, e.g., for the expression of complex biological products such as antibodies or viral vectors. can. Thus, in one embodiment, the expression system comprises one or more additional expression cassettes to enable expression of additional RNAs or proteins of interest. Additional RNAs or proteins of interest may be under the control of the same control element or different control elements. The further expression cassette(s) may contain the same or different promoters and/or the same or different pA signals.

[00106] In some embodiments, the expression systems of this disclosure encode one or more nucleic acids or proteins involved in the production of viral vectors. The term "viral vector" as used herein is intended to include viral particles or virus-like particles capable of transducing target cells. Common viral vectors include, but are not limited to, lentiviral vectors derived from HIV, retroviral vectors, adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. Other viral vectors may be derived from rhabdoviruses (such as vesicular stomatitis virus (VSV)) or herpesviruses (such as CMV and HSV-1). Thus, in one embodiment, the expression system encodes a component of a viral vector. Typical components are structural components of the vector, such as proteins that form the vector's capsid and envelope. Other components are enzymes involved in the replication of vector RNA or DNA. Such enzymes are also involved in the synthesis, maturation, and transport of viral RNA. These enzymes may also be involved in the processing and maturation of viral components and the integration of the viral genome into the cell chromosome. Enzymes that are components of viral vectors may also be involved in the reverse transcription of viral genomic RNA into DNA. Other components of the vector may be proteins or peptides that modulate the replication, transcription, transport or translation of the viral vector's genes or gene products. Such factors can also activate or decrease the expression of cellular genes and can modulate cellular defense mechanisms against viruses. Several viral vector components are toxic to cells, such as adenoviral and lentiviral proteases (encoded by the lentiviral gag/pol genes), AAV Rep protein, and VSV envelope glycoprotein (VSVg). It is well known that it shows This list is not exhaustive, and viral vectors or other components of the virus can be toxic if produced constitutively or in excessively high concentrations.

[00107] In one embodiment, the viral vector is a lentiviral vector. Expression of envelope proteins such as REV, Gag/Pol and VSVg is involved in the production of lentiviral vectors. Accordingly, in some embodiments, the expression system comprises an additional expression cassette encoding each of the envelope proteins, such as REV, Gag/Pol, and VSVg, operably linked to a promoter and at least one viral construct. The element is under the control of a coumermycin inducible promoter. Optionally, all viral components are under the control of a coumermycin inducible promoter. In some embodiments, Gag/Pol is under the control of a constitutive promoter. In some embodiments, Gag/Pol is under the control of a coumermycin inducible promoter.

[00108] In one embodiment, the viral vector is a recombinant adeno-associated virus (AAV). Expression of Rep protein is involved in AAV production. Thus, in some embodiments, the inducible expression system comprises an expression cassette comprising a nucleic acid molecule encoding Rep40, Rep52, Rep68 or Rep78 operably linked to a coumermycin inducible promoter. In some embodiments, the expression system comprises one or more additional expression cassettes encoding Rep40, Rep52, Rep68 or Rep78 operably linked to a promoter, wherein at least one is coumermycin inducible. Under the control of a promoter. In some embodiments, the expression system comprises an expression cassette encoding at least one of Rep40 or Rep52 and an expression cassette encoding at least one of Rep68 or Rep78, wherein at least one is under the control of a coumermycin-inducible promoter. For example, the expression system can include Rep40 and Rep68, Rep40 and Rep78, Rep52 and Rep68, or Rep52 and Rep78. Optionally, all viral components are under the control of a coumermycin inducible promoter.

[00109] In some embodiments, the gene expression system encodes an antibody fragment, an antibody heavy chain, and/or an antibody light chain. The antibody fragment, antibody heavy chain and/or antibody light chain may be encoded in separate expression cassettes, at least one of which is under the control of a coumermycin-inducible promoter.

[00110] As used herein, the term "antibody" is intended to include monoclonal, polyclonal, chimeric and humanized antibodies. Antibodies may be of recombinant origin and/or produced in transgenic animals. As used herein, the term "antibody fragment" refers to Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies and multimers thereof, It is intended to include, but not be limited to, specific antibody fragments, as well as domain antibodies. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can be expressed as recombinant proteins. can.

[00111] The basic antibody structural unit is known to include a tetramer composed of two identical pairs of polypeptide chains, each pair containing one light chain (“L”) (approximately 25 kDa) and one heavy chain (“H”) (approximately 50-70 kDa). The amino-terminal portion of the light chain forms the light chain variable domain (VL) and the amino-terminal portion of the heavy chain forms the heavy chain variable domain (VH). The VH and VL domains together form the antibody variable region (Fv), which is primarily responsible for antigen recognition/binding. The carboxy-terminal portions of the heavy and light chains together form a constant region that is primarily responsible for effector functions.

III. Method
[00112] The expression systems described herein encoding RNA or proteins of interest are introduced into mammalian cells for inducible production of one or more RNAs or proteins of interest encoded therein. It is possible. Accordingly, one aspect of the present disclosure is a method of producing a mammalian cell for the inducible production of one or more RNA or proteins of interest, the method comprising: by introducing an expression system described herein into a mammalian cell, introducing a selectable marker into the cell, and applying selective pressure to the cell to select for cells bearing the selectable marker; including selecting cells harboring the expression system.

[00113] A variety of mammalian cells can be used for production of the RNA or proteins of interest. Suitable cells are well known in the art and include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, VERO cells, HeLa cells, A549 cells, stem cells, and May contain neurons. In some embodiments, the cells are HEK293 cells, optionally 293SF-3F6 cells as described in US Pat. No. 6,210,922. In some embodiments, cells can be grown in suspension and/or grown in the absence of serum.

[00114] The expression system can be introduced into cells by any suitable method known in the art. Suitable methods include, but are not limited to, transfection, transduction, infection, electroporation, sonoporation, nucleofection, and microinjection. In some embodiments, the nucleic acid construct is introduced into the cell by transfection. Suitable transfection reagents are well known in the art and include cationic polymers such as polyethyleneimine (PEI), cationic lipids such as Lipofectamine and related reagents (Invitrogen), and non-transfection agents such as Fugene and related reagents (Promega). Liposomal reagents or calcium phosphate may be mentioned. In some embodiments, expression systems can be introduced into cells by transduction using appropriate viral vectors such as lentiviruses, retroviruses, AAV, and adenoviruses.

[00115] A selectable marker is introduced into the cells together with the expression system or with one or more expression cassettes of the expression system to enable selection of cells into which the expression system or its components have been introduced. Good too. As used herein, the term "selectable marker" refers to an element in a nucleic acid construct that confers a selective advantage to cells harboring the nucleic acid construct. For example, a selectable marker may encode a protein that is expressed and confers resistance to a particular drug. Alternatively, the selectable marker may encode a protein that is expressed and essential for cell viability under particular growth conditions. Appropriate selectable markers are known to those skilled in the art. Examples of suitable drug selectable markers include blasticidin resistance, neomycin resistance, hygromycin resistance, or puromycin resistance.

[00116] The selectable marker may be on the same nucleic acid molecule as the expression cassette or on a different nucleic acid molecule. If the selectable marker is on another nucleic acid molecule, the nucleic acid molecule with the selectable marker is in a lower ratio or percentage than the other nucleic acid molecule(s), e.g., 1:4 molar ratio, 1:5 molar ratio. , or in a 1:10 molar ratio, or for example 25%, 20%, or 10% of the total nucleic acids. Cells that have taken up the selectable marker are likely to have taken up other nucleic acids that are introduced along with the marker, such as the expression system or its components, thereby allowing selection of cells that carry the expression system or its components. .

[00117] Stable cell lines containing one or more expression cassettes of an expression system are obtained by isolating individual cells containing one or more expression cassettes and culturing the cells to produce a stable cell line containing one or more expression cassettes. It can be produced by producing a cell population containing the following. Accordingly, in one embodiment, the method further comprises isolating individual cells and culturing the individual cells to create a cell population.

[00118] One or more expression cassettes of an expression system can be introduced into a cell simultaneously and/or sequentially. As an example, all three expression cassettes may be introduced into a cell in a single step (eg, a single transfection or a single transduction). Alternatively, the first and second expression cassettes are introduced into the cell in a single step, and the third expression cassette and optionally the fourth and/or fifth expression cassette are introduced into the cell in one or more subsequent steps. may be introduced. Alternatively, each expression cassette can be introduced into cells sequentially. Accordingly, in one embodiment, a method of producing a mammalian cell for producing an RNA or protein of interest comprises the steps of: introducing a first expression cassette of an expression system and a first selectable marker into the mammalian cell; selecting cells carrying the first expression cassette by applying selective pressure on the cells to select cells carrying the first selectable marker; isolating the cells; culturing the first individual cells to obtain a first cell population comprising the first expression cassette; injecting the cells of the first cell population with a second expression system; introducing the cassette and a second selectable marker; selecting cells carrying the second expression cassette by applying selective pressure on the cells to select cells carrying the second selectable marker; isolating a second individual cell having the second expression cassette; culturing the second individual cell to obtain a second population of cells comprising the second expression cassette; introducing a third expression cassette and a third selectable marker into cells of the second cell population; applying selective pressure on the cells to select cells carrying the third selectable marker; selecting cells carrying the third expression cassette; isolating a third individual cell carrying the third expression cassette; and culturing the third individual cells to obtain the third expression cassette. obtaining a third cell population having .

[00119] Mammalian cells containing the expression systems described herein can be used for the inducible production of complex biological products such as antibodies or viral vectors. Accordingly, in one embodiment, the method further comprises introducing into the cell one or more additional expression cassettes encoding additional components. Additional expression cassettes can be introduced into the cell, along with additional selectable markers, and/or any other expression cassettes, and/or any other additional components. For example, if the expression system is used for inducible production of viral vectors, additional components may include a viral construct carrying the gene of interest (encoding RNA or protein).

[00120] The expression systems described herein can be used for inducible production of one or more RNAs or proteins of interest encoded therein. Accordingly, one aspect of the present disclosure is a method of inducing the production of one or more RNA or proteins of interest, comprising: a) obtaining a mammalian cell comprising an expression system of the present disclosure encoding an RNA protein of interest; b) adding an inducing agent to the growth medium of the cells to induce expression from an inducible promoter; and c) culturing the cells under conditions for production of the RNA or protein of interest; This method includes the step of producing the desired RNA or protein by.

[00121] The expression system includes a coumate-inducible promoter and a coumermycin-inducible promoter. Accordingly, suitable inducers include coumate effector molecules and coumermycin effector molecules. In one embodiment, the cumate effector molecule is cumate. Any suitable concentration of cumate may be used, for example about 1-200 μg/ml, about 50-150 μg/ml, or optionally about 100 μg/ml. In one embodiment, the coumermycin effector molecule is coumermycin. Any suitable concentration of coumermycin may be used, eg, about 1-30 nM, about 5-20 nM, or optionally about 10 nM. The cumate effector molecule and the coumermycin effector molecule can be added to the growth medium at about the same time or sequentially.

IV. Composition
[00122] The expression systems of this disclosure include three or more expression cassettes, each comprising a nucleic acid molecule. The expression cassettes described herein can be provided as one or more nucleic acid molecules or constructs. It is understood that the nucleic acid molecule or construct may be integrated into the genetic material of a cell or integrated into a plasmid. Thus, in one aspect, the one or more expression cassettes of the expression systems described herein are in the form of one or more plasmids containing the expression cassette(s) of the expression system; may be provided in the form of a cell containing one or more expression cassettes. In one embodiment, one or more expression cassettes are provided on one or more plasmids. In one embodiment, one or more expression cassettes may be integrated into the genetic material of the cell.

[00123] Another aspect of the disclosure includes mammalian cells useful for inducible expression of RNA or proteins of interest. Accordingly, in one embodiment, the present disclosure provides a mammalian cell comprising an expression system described herein that encodes an RNA or protein of interest. As used herein, "cell containing an expression cassette," "cell containing an expression system," or similar phrases refers to a cell into which the indicated expression cassette or expression system nucleic acid molecule(s) have been introduced. means. Suitable methods for introducing nucleic acid molecules into cells are well known in the art.

[0124] In one embodiment, mammalian cells are useful for inducible production of viral vectors. Thus, in one embodiment, the mammalian cell contains an additional expression cassette encoding one or more components of the viral vector. In one embodiment, the viral vector is a lentiviral vector. In one embodiment, the viral vector is an adeno-associated virus (AAV).

[00125] In one embodiment, the mammalian cell is a viral packaging cell that contains an expression cassette encoding a component of a viral vector. In one embodiment, the viral packaging cell comprises an expression cassette encoding a viral envelope protein, such as the lentivirus REV, Gag/pol, and/or VSVg, optionally VSVg-Q96H-I57L. It is. In one embodiment, the viral packaging cell comprises an AAV expression cassette encoding, for example, Rep40, Rep52, Rep68, Rep78, or a combination of at least one of Rep40 or Rep52 and at least one of Rep68 or Rep78. It is a packaging cell. For example, the expression system can include Rep40 and Rep68, Rep40 and Rep78, Rep52 and Rep68, or Rep52 and Rep78.

[00126] Viral constructs are made from DNA or RNA and include a portion of the genetic material of the virus from which they are derived, such as lentiviruses, retroviruses, AAV and adenoviruses. Viral constructs are modified to carry and deliver genes of interest to produce recombinant proteins or RNA of interest and can be used, for example, in the treatment of diseases by cell and gene therapy and in vaccination. Viral constructs can also be used to introduce genes of interest to produce recombinant proteins or RNA in cell culture. Appropriate viral constructs are well known in the art and will vary depending on the viral vector and type of virus used. Thus, in one embodiment, the viral packaging cell further comprises a viral construct carrying the gene of interest. The type of viral construct depends on the viral packaging cell (or viral vector) used. For example, if the viral packaging cell is a lentiviral packaging cell, the viral construct is a lentiviral construct. When the viral packaging cell is an AAV packaging cell, the viral construct is an AAV construct.

[0127] In one embodiment, mammalian cells are useful for the inducible production of antibodies. Thus, in one embodiment, the mammalian cell contains an expression cassette encoding one or more additional components of the antibody.

[00128] To provide a flexible tool for customizable expression of one or more RNAs or proteins of interest, mammalian cells may contain only some of the expression systems described herein. It's okay to stay. For example, the mammalian cell may be an "expression-ready" cell that includes the first and second expression cassettes of the expression systems described herein. The third expression cassette is incorporated, for example, into a plasmid that can be customized by the user to encode the RNA or protein of interest and is inserted into expression-prepared cells to create cells for inducible production of the RNA or protein of interest. can be introduced.

V. kit
[00129] The expression systems described herein can be provided as kits for inducible expression of RNA or proteins of interest. Accordingly, one embodiment includes a kit for inducible expression of RNA or protein of interest. In one embodiment, the kit includes a plasmid encoding an expression system of the present disclosure. In another embodiment, a kit includes cells containing an expression system of the present disclosure and one or more inducing agents. In a further embodiment, the kit comprises "expression ready" cells comprising the first and second expression cassettes of the expression system and a plasmid comprising a coumermycin inducible promoter, a cloning site and a polyadenylation signal and/or a third expression cassette. Contains a plasmid containing.

[00130] If the expression system produces a viral vector, the kit can include a viral packaging cell containing an expression system of the disclosure encoding components of the viral vector, and the appropriate viral construct.

[00131] The following non-limiting examples illustrate the present disclosure:
VI. Example
[00132] In this disclosure, cell lines, such as cell lines derived from human embryonic kidney cells (HEK293 cells), are synthesized with a repressor of the cumate gene switch (known as CymR) and a chimeric transactivator of coumermycin (λR). - GyrB). HEK293-derived 293SF-3F6 cells were used herein because they are grown in suspension culture in serum-free media for ease of scale-up and regulatory compliance. The resulting cell line is called 293SF-CymR/λR-GyrB (Figure 1).

[0133] The level of induction brought about by the cumate/coumermycin gene switch, as determined by comparing the On/Off ratio of gene expression before (Off) and after induction (On), 130-fold higher compared to the gene switch and 30-fold higher compared to the cumate gene switch in the reverse transactivator configuration (Figure 5).

[00134] Herein, we demonstrate the utility of a novel cumate/coumermycin gene switch for the production of complex biological products by use in the generation of packaging and production cells for lentivirus (LV)-derived viral vectors. Show your gender. LV is a very important vector in gene therapy and cell therapy applications (Dropulic, 2011; Escors and Breckpot, 2010; Matrai et al.). The LV packaging and production cells described herein are derived from 293SF-CymR/λR-GyrB cells and are capable of suspension culture and growth in serum-free media. To produce LV, cells must produce cytotoxic proteins such as the envelope glycoprotein of vesicular stomatitis virus (VSVg) and the protease encoded by the Gag/pol gene of human immunodeficiency virus (HIV). be. Cells containing these genetic elements can only be constructed using very tightly controlled inducible gene expression systems.

[0135] Also described herein is the production of adeno-associated virus (AAV) packaging cells. AAV is another important viral vector for gene and cell therapy applications (Balakrishnan and Jayandharan, 2014; Kotterman and Schaffer, 2014; Robert et al., 2017; Weitzman and Lind en, 2011). More specifically, the use of the coumate/coumermycin gene switch to construct a cell line (293SF-Rep) expressing the highly cytotoxic Rep protein of AAV is demonstrated herein. Rep protein is essential for AAV replication and assembly. As shown herein, 293SF-Rep cells were able to produce AAV after induction with cumate and coumermycin.

Example 1. Construction of 293SF-CymR/λR-GyrB
[00136] The first step to generate the 293SF-CymR/λR-GyrB cell line was to construct a stable cell line (293SF-CymR) expressing a repressor of the cumate gene switch.

Construction of 293SF-CymR cell line (clone 198-2)
[00137] A clone of HEK293 cells adapted to serum-free suspension culture (clone 293SF-3F6 (Cote et al., 1998)) was used as a recipient of the CymR gene. Briefly, the cells were transfected with a plasmid encoding the CymR gene controlled by the CMV5 promoter (Fig. 2A) (an example of the first expression cassette disclosed herein) and a plasmid encoding puromycin resistance. did. After transfection, cells were diluted in the presence of puromycin in 96-well plates. Resistant colonies were picked and amplified. The presence of CymR in the clones was tested by transducing them with LV expressing GFP controlled by the CMV5CuO promoter (LV-CMV5CuO-GFP, Figure 3A). GFP expression levels after transduction with LV-CMV5CuO-GFP and induction with cumate were visualized by fluorescence microscopy or quantified by comparing induced and uninduced cells by flow cytometry (On/ Off ratio). Clones with the best On/Off ratio were expanded and banked (a subset of the cell population that was neither induced nor LV treated was used for cell expansion and cell banking). Next, the clones with the best On/Off ratios (#169 and #198) were subcloned by plating at low cell density in a semi-solid medium. Well-separated colonies were then picked using a robotic cell picker (ALS CellCellector™) as previously described (Caron et al., 2009). The ability of CymR to regulate gene expression was tested by amplifying subclones and transducing cells with LV-CMV5CuO-GFP. One of the best subclones is called clone 198-2.

Insertion of λR-GyrB into 293-CymR cells
[00138] A plasmid encoding the λR-GyrB transactivator controlled by the CMV5CuO promoter (Figure 2B) (an example of the second expression cassette disclosed herein) was introduced into 293SF-CymR cells (clone 198-2). ) and a plasmid encoding blasticidin resistance. After transfection, cells were plated in 96-well plates in the presence of blasticidin. Resistant colonies were picked, amplified, and tested for the presence of λR-GyrB by transducing LV expressing GFP controlled by the 12xlambda-TPL promoter (LV-12xlambda-TPL-GFP, Figure 3B). After induction with coumate and coumermycin, GFP expression was measured by flow cytometry and the best clone was selected based on the highest On/Off ratio. The best clones were amplified and banked as above. Subcloning was performed by limiting dilution in a 96-well plate. Colonies were picked, expanded, transduced with LV-12xlambda-TPL-GFP, and tested for the presence of λR-GyrB as described above by analysis by flow cytometry after induction.
The cumate/coumermycin gene switch provides better induction levels than the cumate gene switch

[00139] Efficacy of the cumate/coumermycin gene switch by testing the On/Off ratio of three of the best clones of 293SF-CymR/λR-GyrB after transduction with LV-12xlambda-TPL-GFP. was evaluated. The On/Off ratios obtained with the best clone of 293SF-CymR (described above) and the best clone of 293SF-CymR/rcTA were also tested for comparison. The 293SF-CymR/rcTA clone was obtained by transfecting 293SF-3F6 cells with a plasmid encoding the CMV-regulated CymR and a plasmid encoding the reverse transactivator rcTA controlled by the CMV5CuO promoter (Fig. 2C), and as described above. It was created by isolating a clone in which both genes were stably integrated into the chromosome. In the case of 293SF-CymR/rcTA, addition of cumate activates transcription of the rcTA gene (by releasing inhibition by CymR). After synthesis, rcTA binds to the CR5 promoter and activates transcription (Mullick et al., 2006) (Figure 4). A detailed description of the steps used to construct the 293SF-CymR/rcTA clone is provided in the section entitled Materials and Methods.

[00140] The On/Off ratio of 293SF-CymR and 293SF-CymR/rcTA clones were determined by transducing cells with LV-CMV5CuO-GFP and LV-CR5-GFP, respectively, and measuring the GFP expression level by flow cytometry after induction. It was tested by LV-CR5-GFP carries the GFP gene controlled by the CR5 promoter (Fig. 3C). The observed On/Off ratios were approximately 30, 100, and 4000 for the 293SF-CymR, 293SF-CymR/rcTA, and 293SF-CymR/λR-GyrB clones, respectively (Figure 5). Cells expressing CymR/λR-GyrB had an On/Off ratio of 130 times (4000/30) compared to cells expressing CymR alone or the CymR/rcTA combination. ) and 40 times (4000/100) higher, indicating that the cumate/coumermycin switch provides much better induction levels.

Example 2. Construction of packaging cells for LV production
[00141] As one example demonstrating the utility of the cumate/coumermycin gene switch for producing complex biological products, one of the 293SF-CymR/λR-GyrB clones was used to produce LV. An inducible packaging cell line was constructed. LVs are critical for cell therapy as they are used to genetically modify cells that are delivered to patients to treat cancer or genetic diseases (Dropulic, 2011; Milone and O'Doherty, 2018 ). One of the challenges in the field of cell therapy is the timely production of good quality LV in the quantities required for clinical applications at a reasonable cost.

[00142] One solution to facilitate the production of LVs is to construct packaging cells that contain all the genetic elements necessary for LV assembly. For third generation LV, three genes are required to produce LV: Rev, Gag/Pol and envelope protein (Cockrell and Kafri, 2007; Dropulic, 2011; Pluta and Kacprzak, 2009). The most commonly used envelope protein is VSVg. This availability of packaging cell lines allows scientists to create stable producer clones capable of producing LVs without the need for transfection. The advantages of production clones over transient transfections are reproducibility and simplicity (no transfection and plasmid preparation required). Utilizing packaging cells adapted for suspension culture greatly facilitates scale-up of LV production. In addition, the use of serum-free media results in a safer and better characterized product, which facilitates cGMP production.

[00143] Because some of the genes required to produce LV (Gag/pol and VSVg) are cytotoxic, strict precautions must be taken during cell growth and cell banking to avoid killing host cells. Must be turned off. Therefore, successful construction of LV packaging cells was only possible by using efficient inducible expression systems (Broussau et al., 2008; Farson et al., 2001; Kafri et al., 1999; Ni et al., 2005; Pacchia et al., 2001; Sanber et al., 2015; Sparacio et al., 2001).

[00144] The first step to create packaging cells was to construct a plasmid carrying the Rev, Gag/pol and VSVg genes. First, a plasmid was constructed in which transcription of Rev and VSVg was controlled by the 13xlambda-TPL promoter (FIGS. 6 and 15). For Gag/Pol expression, two different promoters were tested: 11xlambda-hbgmin and the CAG promoter (Figures 6 and 16). CAG is a strong constitutive hybrid promoter that fuses the CMV enhancer to the actin promoter (Miyazaki et al., 1989). In the latter case, even though Gag/pol is controlled by a strong constitutive CAG promoter, in the absence of Rev protein its mRNA is not transported into the cytoplasm and translated into polyprotein. Therefore, the production of Gag/pol polypeptide requires the presence of Rev. Therefore, transcription of the Rev gene indirectly controls the synthesis of Gag/pol polypeptides.

[00145] Two strategies were employed to produce packaging cells. In the first strategy, 293SF-CymR/λR-GyrB cells were infected with 11xlambda-hbg-Gag/Pol, 13xlambda-TPL-Rev, 13xlambda-TPL-VSVg (the third, fourth, and fifth expression groups disclosed herein). (an example of a cassette) and a fourth plasmid encoding resistance to neomycin (an example of a selectable marker disclosed herein). In the second strategy, 293SF-CymR/λR-GyrB cells were infected with CAG-Gag/Pol, 13xlambda-TPL-Rev, 13xlambda-TPL-VSVg, and a plasmid encoding resistance to hygromycin (disclosed herein). Other examples of selectable markers).

[00146] After transfection, selection agents were added to the cell culture medium. The pool of resistant cells was diluted and cloned into nanowells. Resistant colonies derived from single cells (photographed and recorded at the time of plating) were isolated using a robotic cell picker (Cell Collector™) and transferred to 384-well plates. Cells were then grown and tested for LV production.

[00147] Clones of packaging cells were cloned with a plasmid encoding a LV expressing GFP controlled by CMV (LV-CMV-GFP) (Figure 3D) (an example of a lentiviral construct disclosed herein). Screened for LV production by transfection. A subpopulation of cells that were not transfected was retained for best clone amplification and cell banking. The titer of LV-CMV-GFP produced after transient transfection was measured by flow cytometry after transduction into HEK293 cells. Both strategies using the CAG-Gag/Pol plasmid or the 11xlambda-hbgmin-Gag/Pol plasmid generate packaging cells with titers greater than ^1.0X106 transducing units (TU) per ml of culture medium. was completed. Some clones also produced titers above 1.0×10 ⁷ TU/ml. (Figure 7). Previous attempts to isolate LV-producing clones using packaging cells constructed solely with the coumate switch were unsuccessful (not shown).
Example 3. Construction of stable producers of LV
[00148] Packaging cells (clone 3D4 (Figure 7B)) were used to generate production clones with the ability to produce LVs without the need for transient transfection. Briefly, packaging cells 3D4 were co-transfected with a plasmid encoding LV-CMV-GFP (Fig. 3D) and a plasmid encoding neomycin resistance. After transfection, a selection agent (neomycin) was added to the cells, and neomycin-resistant colonies were diluted and cloned into nanowell plates. Colonies were isolated using a robotic cell picker (Cell Collector™) and transferred to 384-well plates. Clones were expanded and inducing agents (coumate and coumermycin) were added to test for production of LV-CMV-GFP. After transduction of HEK293 cells, LV was titrated by flow cytometry. Some clones were able to produce LV-CMV-GFP in the range of 1.0×10 ⁸ TU/ml in the culture medium (FIG. 8).

Regulation of gene expression in packaging cells
[00149] To confirm the effectiveness of the cumate/coumermycin gene switch in LV packaging cells, expression of genetic elements (Rev, Gag and VSVg) required for LV production was determined by Western blot before and after induction. Analyzed. As expected, the expression of Rev, Gag and VSVg was strongly induced after the addition of coumate and coumermycin (Fig. 9), with very weak or no expression detected before induction.

Example 4. Construction of packaging cells for AAV production
[00150] One common method for producing adeno-associated virus (AAV)-derived viral vectors is to transiently isolate HEK293 cells with three plasmids carrying the necessary elements to assemble functional AAV particles. by transfection (Balakrishnan and Jayandharan, 2014; Grieger and Samulski, 2012; Robert et al., 2017; Wright, 2009). The plasmids are: i) an expression plasmid carrying the genes (viral construct) delivered by AAV, ii) a helper plasmid encoding essential helper genes derived from adenovirus, and iii) a Rep- plasmid containing the Rep and Cap genes of AAV. Cap plasmid. Rep produces four proteins (Rep40, Rep52, Rep68, and Rep78) that are involved in AAV genome replication and packaging. Cap encodes a structural protein that constitutes the capsid of AAV. One potential approach to facilitate the production of AAV may be to use packaging cells containing the necessary elements for AAV production, as in the case of LV. However, because Rep encodes a cytotoxic protein, it is difficult to generate packaging cells for AAV. As further evidence of the utility and effectiveness of the coumate/coumermycin gene switch, an AAV packaging cell (293SF-Rep) was constructed that produces Rep protein under the control of this gene switch. Production of AAV using 293SF-Rep cells was also demonstrated.

[00151] To construct 293SF-Rep cells, 293SF-CymR/λR-GyrB cells were injected with a plasmid encoding Rep52, a plasmid encoding Rep68, and a Rep78, each regulated by the 13xlambda-TPL promoter (Figure 10A). (Examples of the third, fourth and fifth expression cassettes disclosed herein) and a plasmid encoding hygromycin resistance (Examples of the selectable marker disclosed herein) were transfected. . The Rep40 plasmid was not included in this experiment, as Rep40 is not essential for AAV production in the presence of Rep52 (Chahal et al., 2018). After transfection, hygromycin was added to the culture medium to create a hygromycin-resistant pool, which was cloned by limiting dilution in a 96-well plate. Hygromycin-resistant colonies were isolated and expanded using three plasmids: one for Cap (pCMV-CAP), one for the adenovirus helper gene (pHelper), and an expression plasmid with GFP regulated by the CMV promoter (pAAV-CMV-GFP). AAV production was tested by transient transfection with (Fig. 10b). The amount of AAV produced was measured by transducing HEK293A cells with AAV and scoring the percentage of GFP-positive cells semi-quantitatively using fluorescence microscopy or quantitatively by flow cytometry. Clones capable of producing AAV were amplified and banked (a subpopulation of non-transfected and non-induced cells was used for this purpose). After induction with coumate and coumermycin, production of Rep protein was demonstrated by Western blot for some of these clones (FIG. 11). AAV production from clone 13 was investigated by varying the ratio of plasmids (Figure 12). Under some conditions, clone 13 was able to produce 2.5×10 ⁷ infectious virus particles (IVP) of AAV-CMV-GFP per ml by transient transfection. In the absence of induction, the amount of AAV produced was below the sensitivity of this method.

Materials and methods Plasmid construction
[00152] Plasmids were constructed using standard methods of molecular biology, amplified in E. coli, and then purified by chromatography using a commercially available kit (Qiagen Valencia, CA). After purification, plasmid concentration was measured at 260 nm using a NanoDrop™ spectrophotometer (Thermo Scientific). The integrity of the plasmid was confirmed by restriction enzyme digestion. The plasmids required for this project were generated as described below.

[00153] The sequence of the expression cassette for the blasticidin resistance gene cloned into pBlast: pUC57 was ordered from a gene synthesis company (GenScript).

[00154] pHygro: The sequence of the expression cassette for the hygromycin resistance gene cloned into pUC57 was ordered from a gene synthesis company (GenScript).

[00155] pkCMV5-CuO-mcs; This plasmid was created by removing the Rev gene from pkCMV5-CuO-Rev (Broussau et al., 2008) by digesting it with restriction enzymes.

[00156] pKCMV5-CuO-rcTA-Hygro: The rcTA sequence (Mullick et al., 2006) was removed from pAdenovatorCMV5-CuO-rcTA by BlpI and SwaI digestion, and pKCMV5-CuO-Re was obtained by KpnI (blunt) and BlpI digestion. v (Broussau et al., 2008) to replace the Rev sequence, thereby creating the pKCMV5-CuO-rcTA plasmid. The hygromycin expression cassette was removed from pMPG-CMV5-CymRopt-Hygro (Gilbert et al., 2014) by digestion with NruI and BssHII, the ends filled in, and the pKCMV5- ligated into the CuO-rcTA plasmid.

[00157] pKCMV5-CuO-λR-GyrB: The λR-GyrB sequence was removed from pGyrb (Zhao et al., 2003) by NdeI and DraI digestion and the ends were filled in. A DNA fragment containing the λR-GyrB sequence was used to replace the Rev sequence in pKCMV5-CuO-Rev (Broussau et al., 2008).

[00158] pLVR2-CR5-GFP: pRRL. cppt. CR5-GFP. The LV vector sequence (RSV to GFP) from WPRE (Mullick et al., 2006) was transferred into the LVR2-GFP plasmid (Vigna et al., 2002) by digestion with SphI and SalI.

[00159]9_SG_pMA-12xlambda-CMVmin-Protease: This plasmid was ordered from GeneArt (ThermoFisher). It contains adenoviral protease sequences under the control of the 12xlambda-CMVmin promoter.

[00160] pME_005 (pVV-13xlambda-CMVmin-VSVg-Q96-I57L): The CMV5 promoter of pNN02 was replaced with the 12xlambda-CMVmin fragment amplified from pVR10 (see below). Sequencing revealed that the resulting plasmid had 13 copies of lambdaOP instead of 12.

[00161] pMPG-CMV5-CymR: The hygromycin expression cassette was removed from pMPG-CMV5-CymRopt-Hygro (Gilbert et al., 2014) by NruI/AscI (embedding) digestion, and the plasmid was ligated and recircularized. .

[00162] pMPG-Puro: The puromycin expression cassette was isolated from plasmid pTT54 (Poulain et al., 2017) and inserted into a plasmid derived from pMPG (Gervais et al., 1998) without an insert.

[00163] pNN02 (pVV-CMV5-VSVg-Q96-I57L): To construct this plasmid, we first removed the BglII/BbsI fragment containing the DS and FR sequences from the pTT5 vector (Durocher et al., 2002). pVV-CMV5 was created by filling in the ends and recircularizing the plasmid. Next, the VSVg gene (VSVg-Q96-157L shown in SEQ ID NO: 19, codon-optimized based on the amino acid gene bank accession number ABD73123.1 shown in SEQ ID NO: 20) ordered from GenScript was added to pVV-CMV5. It was cloned into the PmeI site of.

[00164] pNRC-LV1 (pNC109): Three DNA fragments containing the complete backbone of the lentiviral vector (see Figure 3, without expression cassette [CMV-GFP]) were ordered from Integrated DNA Technologies. , and then ligated into a pMK (GeneArt™, ThermoFisher)-derived cloning vector by Gibson assembly. The resulting plasmid is called pBV3. To obtain higher titers, the fragment (CMV5'UTR-HIV-1Ψ-RRE-cPPT, Genbank accession number FR822201.1) was ordered from a gene synthesis company (GeneScript) and homologous to pBV3 by XbaI/SalI digestion. used to replace the region.

[00165]pNRC-LV1-CMV-GFPq (pNC111): CMV-GFP was amplified from pCSII-CMV-GFPq (Broussau et al., 2008) by PCR, and introduced into pNRC-LV1 having an Esp3I site by Golden Gate assembly. did.

[00166] pSB178 (pKCR5-VSVg-Q96H-I57L) and pSB174 (pKCR5-Rev) were both modified to replace the CMV-B43 cassette with the CR5 promoter from Kumate Switch (Mullick et al., 2006). It is derived from the pKCMV-B43 vector (Mercille et al., 1999). VSVg-Q96H-I57L (SEQ ID NO: 19, which is codon-optimized based on Amino Acid Gene Bank accession number ABD73123.1 as shown in SEQ ID NO: 20) and Rev (Gene bank accession number AF033819.3 ) sequences were ordered from a gene synthesis company (GenScript) and cloned downstream of the CR5 promoter to create pSB178 and pSB174, respectively.

[00167] pSB189 (pkCMV5-hbgdelta-Gag/pol2): Gal/pol gene (HIV-1 complete genome Gene bank accession number AF033819.3) and part of the intron of human β-globin (Gene bank accession MK476503.1) ) was ordered from GenScript. Both fragments were cloned into a plasmid derived from pKCMV-B43 (Mercille et al., 1999), which was modified by replacing the CMV-B43 cassette with the CMV5 promoter (Massie et al., 1998a). During cloning, part of the intron of CMV5 was replaced with the intron sequence of human β-globin.

[00168] pSB201 (pMPG/TK*/Neo): This plasmid was created by removing the CymR-nls cassette from pMPG/TK*neo/CymR-nls (Mullick et al., 2006) by AscI digestion.

[00169] pSB211 (pCAG-Gag/polIIIb): The Gag/Polllb sequence (GeneBank accession number EU541617.1) was ordered from a gene synthesis company (Genscript), and the CMV-B43 cassette was inserted into the CAG promoter (Blain et al., 2010). ) into a plasmid derived from pKCMV-B43 (Mercille et al., 1999).

[00170] pSB213 (p11xlambda-hbgmin-Gag/polIIIb): 12lambda-hbgmin promoter and intron were extracted from pVR28 by digestion with BamHI (embedded), pSB211 by digestion with XhoI (embedded) and EcoRI (embedded) (pCAG-Gag/polIIIb) was used to replace the CAG promoter. Sequencing of the promoter revealed that one lambdaOp repeat was lost during cloning, leaving 11 (instead of 12) lambdaOp repeats in the promoter. A schematic diagram of the obtained promoter (11xlambda-hbgmin) is shown in FIG.

[00171] pTet07-CMV5-CuO-GFP: The CMV5-CuO sequence was first transferred to pRRL. ctpt. pNEB- by extracting from CMV5-CuO-rcTA (Mullick et al., 2006), digesting with SpeI and BamHI, filling in the ends, and ligating into pNEB193mcs (NEB) previously digested with XbaI and blunted. CMV5-CuO was produced. The CMV5-CuO sequence from pNEB-CMV5-CuO was then ligated into pTet07-CSII-5-GFP (described below) after digesting both DNAs with PacI and BlpI.

[00172] pTet07-CSII-CMV-mcs: Plasmid LVR2-GFP (Vigna et al., 2002) was first modified by inserting a BspEI linker (TCGATCCGCA) at the XhoI site. The 3'LTR containing the Tet07 operator was then removed from this construct by digestion with BspEI and PmeI, and pCSII-CMV-mcs (pre-blunted) pre-digested with BspEI and BsmI (Miyoshi et al., 1998).

[00173] pTet07-CSII-mcs: The CR5 promoter of pTet07-CSII-5-mcs was removed by digestion with PacI and AgeI to create an empty LV scaffold.

[00174] pTet07-CSII-5-mcs and pTet07-CSII-5-GFP: CMV promoter from Tet07-CSII-CMV-mcs and Tet07-CSII-CMV-GFP (Broussau et al., 2008) into CR5 promoter. A PacI site was inserted at the 5' end of the promoter for substitution and easy modification of the promoter in future constructs. Briefly, first, using a plasmid derived from pCSII-CMV mcs (Miyoshi et al., 1998) as a template, the region from the SnabI site (5' end of 5' LTR) to the cppt sequence of the first CMV promoter was A PCR fragment covering was amplified. In the second PCR, pRRL. cppt. CR5-GFP. was performed to amplify the CR5 promoter from WPRE (Mullick et al., 2006). PacI sites were inserted at the 3' end of the first fragment and at the 5' end of the second fragment. The PCR products were annealed and treated with T4 DNA polymerase to generate one fragment that together covered the CMV to mcs portion 5' of the 5'LTR. This fragment was digested with SnabI and AgeI and inserted into Tet07-CSII-CMV-mcs and Tet07-CSII-CMV-GFP plasmids. Sequencing revealed that five of the six CuO copies of the CR5 promoter were deleted during cloning.

[00175] pTet07-CSII-12xlambda-TPL-GFPq: The 12xlambda-TPL-GFPq sequence from pVR9 was first inserted into pUC19 by digestion with BamHI, thereby creating pUC19-12xlambda-TPL-GFPq. Next, the 12xlambda-TPL-GFPq sequence was digested with EcoRI and then ligated to Tet07-CSII-mcs.

[00176] pVR1 (pKC_12xlambda-CuO-TPL-MCs): The 12xlambda-CuO promoter was ordered from GenScript1, and the CMV-CuO promoter region of pKCMV5-CuO-MSC was replaced by digesting with KpnI/AgeI.

[00177] pVR2: (pKC_12xlambda-CuO-msc-PolyA) was created by introducing 12xlambda-TATA-CuO synthesized by GenScript into the Acc65I/BglII site of pKCMV5-CuO-mcs instead of the CMV5-CuO promoter. Created.

[00178] pVR5 (pKC_12xlambda-CuO-TPL-GFPq): The GFP gene obtained by digesting pAd-CMV5-GFPq (Massie et al., 1998b) with BamHI was ligated to pVR1 digested with BglII.

[00179] pVR6 (pKC_12xlambda-CuO-GFPq): Obtained by subcloning GFPq from BamHI-digested pAdCMV5-GFPq (Massie et al., 1998b) into BglII-digested pVR2.

[00180] pVR9 (pKC_12xlambda-TPL-GFPq): The 12xlambda-CMVmin promoter of 9_SG_pMA-12xlambda-CMVmin-Protease was amplified by PCR, inserted into pVR5 previously digested with AgeI/KpnI, and pVR5 Replaced the 12xlambda-CuO fragment of did.

[00181] pVR10 (pKC_12xlambda-CMVmin-GFP): The KpnI-AgeI fragment of pVR6 (containing 12xlambda-CuO) was previously amplified by PCR using 9_SG_pMA-12xlambda-CMVmin-Protease as a template and digested in the same manner. 12xlambda- It was created by substituting the CMVmin fragment.

[00182] pVR17 (pVV-13xlambda-CMVmin-MCS): DNA containing the CMVmin promoter obtained by removing the CMVmin-VSVg fragment from pME_005 by digesting with SalI and HindIII and performing PCR using plasmid pVR10 as a template. obtained by substituting the fragment.

[00183] pVR19 (pVV-13xlambda-TPL-VSVg-Q96H_I57L): pVR17 (pVV-13lambda-CMVmin-mcs) obtained by similarly digesting the SacI/HindIII fragment containing the cDNA of VSVg (TPL-VSVg) from pSB178. subcloning to Obtained by doing.

[00184] pVR21 (pVV-13xlambda-TPL-Rev): The Rev sequence (TPL-Rev) from pSB174 was obtained by subcloning into pVR17 using StuI/NheI restriction sites.

[00185] pVR28 (pK-12xlambda-hbgmin_ex_Gag-Pol): The AflIII/XhoI fragment of pSB189 encoding the CMV promoter/enhancer and TPL was replaced with the AflIII/XhoI fragment containing the 12xlambda promoter and TPL from pVR9.

[00186] pVR41, pVR42, pVR43 and pVR44: Plasmids containing AAV2 genes encoding Rep78, Rep68, Rep52 and Rep40, respectively, under the control of the 13xlambda-TPL promoter. Plasmids were created as follows: Human optimized AAV2 genes encoding Rep78, Rep68, Rep52 and Rep40 were synthesized in GenScript and each was subcloned into the EcoRV site of pUC57 to create 18_SG, 19_SG, 20_SG and 21_SG. . The TATA box of the internal P19 promoter within the Rep68 and Rep78 sequences was modified to reduce its activity by changing the TATTTAAGC sequence to the TACCTCTCA sequence. The pUC57-Rep clone was digested with BglII/NotI, and the fragment encoding the Rep gene was subcloned into similarly digested pVR19 (pVV-13xlambda-TPL-VSVg) instead of VSVg, and pVR41 (pVV-13xlambda-TPL-Rep78 ), pVR42 (pVV-13xlambda-TPL-Rep68), pVR43 (pVV-13xlambda-TPL-Rep52) and pVR44 (pVV-13xlambda-TPL-Rep40) were obtained.

[00187] pVR46-Cap: Encodes the AAV2 CAP gene regulated by the CMV5 promoter. To construct this plasmid, pTT3CAP1 was generated by cloning the AAV2 CAP gene and its upstream untranslated region after the CMV5 promoter of pTT3 (Massie et al, 1998a). The splice acceptor site of the CMV5 promoter and the splice donor site of the Cap gene were removed by digesting with AleI/Swal and ligating the ends.

cell culture
[00188] 293SF-CymR and 293SF-CymR/rcTA were cultured in SFM4-Transfx-293 medium (Hyclone) supplemented with 6mM L-glutamine (Hyclone). Subcloning in semi-solid medium was performed using ClonaCell™ FLEX methylcellulose (StemCell Technology), 2x SFM4-Transfx-293 (Hyclone), 6mM L-glutamine, 2.5% ClonaCell™ ACF C HO replenishment A mixture of materials (StemCell Technology) was used. 293SF-CymR/λR-GyrB was developed in Low-Calcium-SFM medium (LC-SFM) (Gibco) supplemented with 6mM L-glutamine and 10mg/ml rTransferin (Biogems) and suspended in SFM4-Transfx-293. Proliferated. Cell lines 293SF-PacLVIIIB-L and 293SF-LVPIIIIB-GFP were cultured in 50% LC-SFM supplemented with 6mM L-glutamine and 10mg/ml rTransferin and 4mM L-glutamine and 0.1% Kolliphor®. ) was developed in a mixture of 50% Hycell™ TransFx-H (Hyclone) supplemented with 50% Hycell™ TransFx-H (Hyclone) and maintained suspended in 100% Hycell™ TransFx-H. For suspension culture, cells were cultured in shake flasks at 110 rpm. 293A (American Type Culture Collection), 293rtTA (Broussau et al., 2008) and 293rcTA (Mullick et al., 2006) are Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (Hyclone). (Hyclone) . All cell lines were maintained at 37°C in a 5% CO2 humidified atmosphere.

Lentivirus production and titration
[00189] LV-CMV5CuO-GFP, LV-12xlambda-TPL-GFP and LV-CR5-GFP were purified using 293SF-PacLV#29-6 as previously described (Broussau et al), pTet07- CSII-CMV5-CuO-GFP, pTet07-CSII-12xlambda-TPL-GFP and pLVR2-CR5-GFP were transfected under static conditions in LC-SMF + 1% FBS, and 1 μg/mL of doxycycline and 50 μg of cumate were used. /mL. The produced LVs were concentrated by ultracentrifugation on a sucrose cushion (Gilbert et al., 2007), and the suspension was used to transduce fresh 293SF-PacLV #29-6, and the production cell line was isolated for each LV. A pool was created. This pool was suspension amplified in LC-SFM+1% FBS, and LV production was induced by adding doxycycline 1 μg/mL and cumate 50 μg/mL. The produced LVs were harvested after 48 and 72 hours, concentrated by ultracentrifugation on a sucrose cushion, and frozen at -80°C. LV CMV5CuO-GFP, 12xlambda-TPL-GFP, and CR5-GFP were transduced and titrated into 293A, 293rtTA, and 293rcTA cells, respectively, and the percentage of GFP-expressing cells was determined by flow cytometry as previously described. (Broussau et al., 2008).

Preparation of cell line 293SF-CymR
[00190] The 293SF-3F6 cell line (Cote et al., 1998) grown in SFM4-TransFx-293 was transfected with pMPG-CMV5-CymR-opt and pMPG- Puro was transfected using a 9:1 DNA ratio. Both DNAs were previously digested with MfeI. After 48 hours, cells were diluted into 96-well plates at 5,000 cells/well and 10,000 cells/well in SFM4-Transfx-293 medium containing 0.4 μg/ml puromycin (Sigma). Selected cell clones were subcloned by plating on semi-solid medium at 1000 cells/ml and 3000 cells/ml. Colonies were isolated using a robotic Cell Picker Cell Collector™ (ALS, Germany) and transferred to 96-well plates. To screen clones for the presence of CymR, each clone was divided into two populations. One population was used for analysis of GFP expression, and the other population was left intact for clonal expansion and banking. The clones were analyzed for GFP expression after transduction with LV expressing GFP regulated by the CMV5CuO promoter (LV-CMV5CuO-GFP). Transduction was first performed in 96-well plates and cells were induced with cumate (Sigma-Aldrich) at 100 μg/ml. Clones were selected for GFP intensity and then screened a second time for GFP expression under On/Off conditions (with and without inducer).

293SF-CymR-rcTA
[00191] 293SF-3F6 cells were transfected with pMPG-CMV5-CymR-opt plasmid and pMPG-Puro plasmid (9:1 ratio) using Lipofectamine™ 2000 CD. Both DNAs were previously digested with MfeI. After 2 days, cells were diluted to 5000 and 10000 cells per well in a 96-well plate in the presence of 0.4 μg/ml puromycin. Colonies resistant to puromycin were transferred to 48-well or 24-well plates. When the cells reached 50-80% confluence, they were combined into 25 cm ² flasks to form five different pools (C, D, E, F, G). From this point onwards, no puromycin was added to the medium. The pools were mixed to form pool CDEFG.

[00192] Pooled CDEFG was transfected with a 1:1 complex of linearized (XmnI) pkCMV5-CuO-rcTA-Hygro plasmid using PEIpro® (Polyplus Transfection). After 24 hours of culture, the cells were transferred to a medium containing 25 μg/mL hygromycin B (Invitrogen) in a 96-well plate at 1500 cells per well. Colonies were pooled, centrifuged, and resuspended in fresh medium containing 25 μg/mL hygromycin to form different minipools (letters A to H).

[00193] Pools C, F, G and H were plated as fully dispersed cells in semi-solid media. Colonies in semi-solid medium were screened for the presence and level of rcTA by the automated TiSSM method (Transfection in Semi-Solid Medium) and positive colonies were isolated in 96-well plates by Cell Selector™. Briefly, colonies were identified by scanning with Cell Selector™. A 3:1 complex (pkCR5.DsRed) of PEI MAX® (Polysciences) and a reporter plasmid encoding the DsRed fluorescent protein under the control of the CR5 promoter (Clontech laboratories) was added to each colony with a Cell Selector™ robotic arm. I let it settle. At 24 hours post-transfection, a scan was performed to detect basal DsRed fluorescence from colonies (OFF level). A liquid SFM4 medium overlay containing cumate inducer (Sigma-Aldrich, Cat No. 268402, Lot No. 13613HB) at a final concentration of 100 μg/mL was then applied over the semi-solid medium layer and allowed to spread overnight. A second scan was performed 48 hours post-transfection (24 hours post-induction) to measure DsRed fluorescence (ON levels). The OFF and ON images were then compared, colonies with high induction properties were identified, and these colonies were picked out with Cell Selector™ and deposited into individual wells (96-well plate). Clones isolated from TiSSM were gradually transferred to 24-well plates and 25 cm ² flasks and then banked.

293SF-CymR/λR-GyrB
[00194] 293SF-CymR (clone 198-2) was transfected with plasmids pKCMV5-CuO-λR-GyrB and pBlast at a 9:1 DNA ratio by PEI Pro®. The DNA was previously digested with XmnI and XbaI, respectively. Forty-eight hours after transfection, cells were diluted in LC-SFM medium containing 7 μg/ml Blasticidin (Enzo) and transferred to 96-well plates at 1000 cells/well. After one week, the concentration of blasticidin was increased to 10 μg/ml. Selected clones were limited diluted in LC-SFM medium to 0.3 cells/well and 1.0 cells/well in 96 wells and subcloned without selection. To screen clones for their ability to modulate gene expression, each clone was divided into two populations. One population was used for analysis of GFP expression, and the other population was left unmodified for clonal expansion and banking. The clones were introduced with a lentiviral vector expressing 12xlambda-TPL-regulated GFP (LV-12xlambda-TPL-GFP), and then GFP expression was analyzed. Transduction was first performed in 96-well plates, and cells were induced with 100 μg/ml cumate (Sigma-Aldrich) and 10 nM coumermycin (Promega). Clones were selected for GFP intensity and then screened a second time for GFP expression under On/Off conditions (with and without inducer).

[00195] Comparison of inducibility of cumate switch and cumate/coumermycin switch
[00196] The 293SF-CymR, 293SF-CymR/rcTA and 293SF-CymR/λR-GryB clones were transduced with lentiviral vectors in the presence of 8 μg/ml polybrene. 293SF-CymR and 293SF-CymR/rcTA were transduced with LV-CMV5CuO-GFP and LV-CR5-GFP at an MOI of 20 TU, respectively, and cells were induced the next day by addition of 100 μg/ml cumate. The 293SF-CymR/λR-GyrB clone was introduced with LV-12xlambda-TPL-GFP at an MOI of 5 TU, and the next day, 100 μg/ml cumate and 10 nM coumermycin were added to induce the cells. Cells were fixed and processed for flow cytometry analysis 72 hours post-transduction.

Packaging cells for lentiviral vector (293SF-PacLVIIIA)
[00197] 293SF-CymR/λR-GyrB clone 7-2 was cloned into pSB213 (p11xlambda-hbgmin-Gag/polIIIb), pVR19 (pVV-13xlambda-TPL-VSVg) in suspension using PEI Pro®. -Q96H-I57L), pVR21 (pVV-13xlambda-TPL-Rev) and pHygro were transfected at DNA ratios of 40%, 25%, 25% and 10%, respectively. The plasmids were digested with BspHI, ZraI, XmnI, and XmnI, respectively. 36 hours after transfection, cells were diluted to 0.35x10 ⁶ cells/ml in medium containing 65 μg/ml hygromycin. After 4 and 8 days, cells were plated in nanowells (ALS Automated Lab Solutions GmbH, Jena, Germany) at 4.7 cells/nanowell and a hygromycin concentration of 50 μg/ml. Selection in suspension was continued in parallel. 205 (selected for 4 days in suspension) and 171 (selected for 8 days in suspension) resistant colonies were pooled to form two different minipools. This minipool and the pool obtained from the suspension were diluted and cloned into nanowell plates at a cell density of 0.6 cells/nanowell. Isolated cells in nanowells were recorded on day 0 using a Cell Selector™ (robotic cell picker) camera system. A total of 366 colonies were isolated using Cell Selector™ and transferred to 384-well plates.

Packaging cells for lentiviral vector (293SF-PacLVIIIB)
[00198] 293SF -CYMR / λr -Gyrrb clone 7-2, PSB211 (PCAG -GAG / POLIIIB), PVR19 (PVV -13XLAMBDA -TPL -VSVG -VSVG -Q96H -I57L), PVR21 (PVV -13XLAM) BDA -TPL -REV) and pHygro at DNA ratios of 40%, 25%, 25% and 10%, respectively, in suspension with PEI Pro®. The plasmids were digested with BspHI, ZraI, XmnI, and XmnI, respectively. 36 hours after transfection, cells were diluted to 0.5x10 ⁶ cells/ml in medium containing 80 μg/ml hygromycin. After 8 days, cells were plated in nanowell plates at 1.4 cells/nanowell at a hygromycin concentration of 50 or 25 μg/ml. The 173 resistant colonies were pooled to form a minipool. This minipool was cloned after 6 days by dilution into Nanowell plates at a cell density of 0.6 cells/nanowell in medium supplemented with 20 μg/ml hygromycin. Photographs showing the presence of single cells in the nanowells were obtained on day 0 using the Cell Selector™ (robotic cell picker) camera system. 348 colonies were isolated using Cell Selector™ and transferred to a 384-well plate.

Cells producing lentiviral vector (293SF-LVPIIIB-GFP)
[00199] Packaging cells 293SF-PacLVIIIB, clone 3D4 were placed in suspension using PEIpro® with plasmids pNC111 (pNRC-LV1-CMVGFP) and pSB201 (pMPG/TKneo) at a DNA ratio of 4:1. transfected with. The plasmid was previously digested with FspI and XbaI, respectively. 36 hours after transfection, cells were diluted to 0.5x10 ⁶ cells/ml in medium containing 400 μg/ml geneticin (Gibco). After 18 days of selective culture, cells were diluted for cloning into nanowells at a cell density of 0.6 cells/nanowell (without G418). Photographs showing the presence of single cells in the nanowells were obtained on day 0 using the Cell Selector™ camera system. 380 colonies were isolated using Cell Selector™ and transferred to a 384-well plate.

Screening of clones from packaging cells (293SF-PacLVIIIA, 293SF-PacLVIIIB) and production cells (293SF-LVPIIIB-GFP)
[00200] Clones were analyzed for production of lentivirus expressing GFP (LV-CMV-GFP). It was tested first in a 96-well plate, then in a 24-well plate, and finally in a 6-well plate suspension. Clones from packaging cells were transfected with pCSII-CMV-GFP (Broussau et al., 2008) and induced by adding cumate/coumermycin and sodium butyrate. The production clone (293SF-LVPIIIB-GFP) was not transfected and was induced with coumate/coumermycin and sodium butyrate. The produced LV (LV-CMV-GFP) was titrated by transduction of 293A cells, and GFP levels were assessed by fluorescence microscopy for LV produced in 96-well plates and 24-well plates, and in 6-well plates. The LV produced in 2000 was titrated by flow cytometry (Broussau et al., 2008).

Western blotting of Gag/pol, VSVg and REV
[00201] Cells from 293SF-PacLVIIIB, clone 3D4 were tested by Western blot for expression of p24, VSVg and REV proteins. On the day of induction, 25 million cells were centrifuged and resuspended in 25 ml of Hycell™ medium to a final concentration of 1.0×10 ⁶ cells/mL in a 125 ml shake flask. Induction was performed after 1 hour with 80 μg/ml cumate and 10 nM coumermycin. As a negative control, 5 million 293SFCymR/λR-GyRB cells were centrifuged and transferred to -80°C. Eighteen hours after induction, two cell groups were prepared: one to which 8 mM sodium butyrate was added and one to which it was not. 5 ml of cell culture was harvested at 0, 24, 48, and 72 hours after induction. Cells were collected by centrifugation and cell pellets were transferred to -80°C. For Western blot analysis, cells were thawed and lysed with RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% Na deoxycholate). . After 30 minutes of incubation on ice, samples were sonicated and lysates were clarified by centrifugation. Protein concentration was measured with the BIO-RAD DC™ protein Assay (Bio-Rad Laboratories). Equal amounts of total protein were separated on NuPAGE™ 4-12% Bis-Tris Gel (Invitrogen) and tested with anti-HIV1 REV mouse monoclonal antibody (ab85529, abcam), rabbit polyclonal HIV p24 Ab (ProSci catalog #7313), rabbit Polyclonal anti-VSVg tag antibody (ab83196, abcam) followed by horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin (Ig) G antibody or horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin (Ig) G antibody (GE Healthcare UK Limited). It was analyzed by Western blotting. Signals were revealed by chemiluminescence using ECL™ Western Blotting Detection Reagents (Perkin Elmer, Inc) and a digital imaging system (ImageQuant™ LAS 4000 mini biomolecular imaging system). ger, GE Healthcare) .

Generation of 293SF-Rep cells
[00202] pVR43, pVR42, A pool of cells expressing Rep52, 68, 78 was generated by transfecting pVR41 and pUC57-TK-Hygro at rates of 55%, 30%, 15%, and 10%, respectively. Before transfection, the Rep-encoding plasmid was linearized with SpeI and pUC57-TK-Hygro was linearized with XmnI. Forty-eight hours after transfection, cells were centrifuged and resuspended in medium containing hygromycin (40 or 50 μg/ml) to a final concentration of 0.5 x 10 ⁶ cells/ml. Viability and cell proliferation were monitored regularly. A small bank of frozen vials was prepared from a pool of cells cultured for approximately 3 weeks.

[00203] One vial of the cell bank was thawed in Hycell™ medium supplemented with 4mM glutamine and 0.1% Coriphor® without selection. 40 μg/mL selective hygromycin was added 2 days after thawing. Cells were diluted 3-fold in selective medium before subcloning in 96-well plates. Subcloning was performed in two media without selection: Hycell™ supplemented with 4mM glutamine and 0.1% Coriphor®, and HSFM supplemented with 6mM glutamine and 10mg/L transferrin.

[00204] Colonies in 96 wells were transferred to 24 well plates. Once the wells were confluent, one-third of the cell population was transferred to another 24-well plate and tested for AAV production by transient transfection. For transfection, the culture medium was replaced with fresh medium and cells were transfected using 1 μg/ml plasmid and 2 μg/ml PEI Pro®. Transfection was performed using a mixture of ptt3CAP1, pHelper (CellBiolab), and pAAV-GFP (CellBiolab). Induction was performed 4 hours after transfection with 50 μg/ml cumate and 10 nM coumermycin. Harvesting was performed 72 hours after transfection and titrations were performed using a standard AAV gene transfer assay (described below) in 293SF-3F6 cells using BalanCD® medium. Clones capable of producing AAV were amplified and vials of frozen cells were prepared.

Western blot analysis of Rep expression
[00205] Cells of 293SF-Rep clones (#13, 18, 35 and 36) were cultured in Hycell™ medium supplemented with 40 μg/ml hygromycin. Clones were tested by Western blot for the presence of REP protein after no induction or induction with different concentrations of cumate and coumermycin. 2.5 million cells were centrifuged and resuspended in 2.5 ml of fresh Hycell™ medium at a concentration of 1.0 x 10 6 cells/mL in a 6-well plate. Cells were induced with cumate (μg/ml) and coumermycin (nM) at the following concentrations: 0.5 μg/ml/1 nM; 5 μg/ml/1 nM; or 50 μg/ml/10 nM. Cell cultures were harvested by centrifugation 72 hours after induction, and cell pellets were transferred to -80°C. Cell pellets were thawed and lysed using 300 μl of RIPA (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% Na deoxycholate). After incubation for 30 minutes on ice, samples were sonicated and lysates were clarified by centrifugation. Protein concentration was measured with the BIO-RAD DC™ protein Assay (Bio-Rad Laboratories). Equal amounts of total protein (30 μg) were run on NuPAGE™ 4-12% Bis-Tris Gel (Invitrogen) and purified with mouse monoclonal IgG1 anti-REP AAV (ARP American Research Products, Inc. Cat. No. 03-61069). This was then analyzed by Western blotting using a horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin (Ig) G antibody (GE Healthcare UK Limited). Signals were revealed by chemiluminescence using ECL™ Western Blotting detection reagents (Perkin Elmer, Inc) and analyzed with a digital imaging system (Image Cant™ LAS 4000 mini biomolecular imager, GE Healthcare).

AAV production and gene transfer assay using 29SF-Rep cells
[00206] 293SF-Rep cells (clone 13) were centrifuged (300 x g, 5 min) to remove hygromycin (used to maintain clonal selection) and Hycell ( (Trademark) TransFx-H (Hyclone) to a final density of 1.0×10 ⁶ cells/mL in a 6-well plate. Cell suspensions were transfected with 1 μg/mL DNA (using Cell Biolabs' pAAV-GFP and pHelper and pVR46-Cap in different ratios) and 2 μg/mL PEI Pro® (Polyplus). (FIG. 12), and after 4 hours of incubation, the inducer was added. A plasmid mixture with both inducers was prepared and added to the transfected cells at a final concentration of 10 nM coumermycin and 50 μg/mL coumamate. After incubation for 3 days, the induced cells were incubated with a final concentration of 2 mM MgCl2 (Sigma-Aldrich), 0.1% Triton™ X-100 (Sigma-Aldrich), 2.5 U/mL. Benzonese (MilliporeSigma) lysis solution. Two hours after lysis, MgSO4 was added to a final concentration of 37.5 mM to stabilize the virus particles. The lysed cells were centrifuged at 13,000 rpm for 3 minutes, and the supernatant was collected and frozen. The titers of AAV produced were determined using cells in Balan CD® HEK293 medium (FUJIFILM Irvine Scientific). 293SF-Rep cells were plated at 0.5 x ¹⁰ cells/mL in 12-well plates and incubated with a recombinant adenoviral vector encoding luciferase (HD-LUC, ΔE1, ΔE3) (Umana et al. 2001) at an MOI of 5. infected with. Cell lysates containing AAV were diluted from 1:10 to 1:300 and added to infected cells. After 24 hours of incubation, total cell density and viability were recorded, and 293SF-Rep cells were fixed with 2% formaldehyde (Polysciences Inc.) and analyzed on a flow cytometer (BD LSRFortessa™). . Using the GFP% of single cells (10,000 events), the titer of infectious virus particles (IVP)/mL was calculated considering the dilution factor.

[00207] Although this application has been described with reference to examples, the claims should not be limited by the embodiments described in the examples, but should be interpreted in the broadest manner consistent with this specification as a whole. Please understand that it should be given to you.

[00151] All publications, patents and patent applications are incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. is incorporated herein in its entirety. If a term in this application is found to be defined differently in a document incorporated herein by reference, the definition provided herein will serve as the definition for that term.

Sequence list
CymR gene (SEQ ID NO: 1) Pseudomonas putida

CymR protein (SEQ ID NO: 2) Pseudomonas putida
MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAGVSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLDDDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLIFNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR*

CuO (P2) Pseudomonas putida derived from CMV5-CuO (SEQ ID NO: 3)
AACAAACAGACAATCTGGTCTGTTTGTA

CuO(P1) (SEQ ID NO: 4) Pseudomonas putida
AGAAACAAACCAACCTGTCTGTATTA

CMV5-CuO (SEQ ID NO: 5) synthesis

1xlambdaOP (SEQ ID NO: 6) bacteriophage
TCGAGTTTACCTCTGGCGGTGATAG

12xlambdaOP (SEQ ID NO: 7) synthesis
TCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGT TTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAG

13xlambdaOP (SEQ ID NO: 8) synthesis
TCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGT TTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAG

12xlambdaCMVmin (SEQ ID NO: 9) synthesis
TCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGT TTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGACTCTAGATAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCT

13xlambdaCMVmin (SEQ ID NO: 10) synthesis
TCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGT TTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGAGTTTACCTCTGGCGGTGATAGTCGACTCTAGATAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCT

13xlambda-TPL (SEQ ID NO: 11) synthesis

11xlambda-hbgmin (SEQ ID NO: 12) synthesis

λR-GyrB gene (SEQ ID NO: 13) synthesis

λR-GyrB protein (SEQ ID NO: 14) synthesis

C-terminal part of p65 subunit of mouse NF-κB (SEQ ID NO: 15)
PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISS

Rabbit β-globin polyA (SEQ ID NO: 16)
AATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCA

bGH (bovine growth hormone) polyA (SEQ ID NO: 17)
CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGTGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

SV40 poly(A) signal (SEQ ID NO: 18) Simian virus 40
AACTTGTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

Codon-optimized VSVg-Q96-157L (SEQ ID NO: 19) synthesis

VSVg-Q96-157L amino acid (SEQ ID NO: 20) Vesicular stomatitis virus
MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSE DGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQL PDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*

References
Balakrishnan, B., and GR Jayandharan.2014. Basic biology of adeno-associated virus (AAV) vectors used in gene therapy.Curr. Gene Ther. 14:86-100.
Blain, M., Y. Zeng, M. Bendjelloul, PLHallauer, A. Kumar, KE Hastings, G. Karpati, B. Massie, and R. Gilbert. 2010.Strong muscle-specific regulatory cassettes based on multiple copies of thehuman slow Troponin I gene upstream enhancer. Hum. Gene Ther. 21:127-134.
Broussau, S., N. Jabbour, G. Lachapelle, Y. Durocher, R. Tom, J. Transfiguracion, R. Gilbert, and B. Massie. 2008. Inducible Packaging Cells for Large-scale Production of Lentiviral Vectors inSerum-free Suspension Culture. Mol. Ther. 16:500-507.
Caron, AW, C. Nicolas, B. Gaillet, I. Ba,M. Pinard, A. Garnier, B. Massie, and R. Gilbert. 2009. Fluorescent labeling insemi-solid medium for selection of mammalian cells secreting high-levels ofrecombinant proteins. BMC. Biotechnol. 9:42.
Chahal, P., SM Elahi, P. O'Neal, MHVenne, N. Nazemi-Moghaddam, and R. Gilbert. 2018. Key Rep-proteins necessaryfor the adeno-associated virus production by transient trasfection in HEK293cells in suspension and serum- free medium. In 21st Annual Meeting, American Society of Gene and Cell Therapy, May 16-19, 2018, Chicago, IL.
Cockrell, AS, and T. Kafri. 2007. Genedelivery by lentivirus vectors. Mol Biotechnol. 36:184-204.
Cote, J., A. Garnier, B. Massie, and A. Kamen. 1998. Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells. Biotechnol. Bioeng. 59:567-575.
Dropulic, B. 2011. Lentiviral vectors: their molecular design, safety, and use in laboratory and preclinical research.Hum. Gene Ther. 22:649-657.
Durocher, Y., S. Perret, and A. Kamen.2002. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 30:E9.
Escors, D., and K. Breckpot. 2010. Lentiviralvectors in gene therapy: their current status and future potential. Arch.Immunol. Ther. Exp. (Warsz. ). 58:107-119.
Farson, D., R. Witt, R. McGuinness, T. Dull, M. Kelly, J. Song, R. Radeke, A. Bukovsky, A. Consiglio, and L. Naldini.2001. A new-generation stable inducible Cell packaging line for lentiviralvectors. Hum. Gene Ther. 12:981-997.
Gervais, C., D. Paquette, A. Burns-Tardis,L. Martin, and B. Massie. 1998. Development of high output expression vectorsfor antibody production in mammalian cells. In Animal Cell Technology: Basic& Applied Aspects. K. Nagai and M. Wachi, editors. Kluwer AcademicPublishers, Netherlands. 349-354.
Gilbert, R., S. Broussau, and B. Massie.2007. Protein production using lentiviral vectors. In Methods Express:expression systems. MR Dyson and Y. Durocher, editors. Scion Publishing Ltd,Oxfordshire. 241-258.
Gilbert, R., C. Guilbault, D. Gagnon, A.Bernier, L. Bourget, SM Elahi, A. Kamen, and B. Massie. 2014. Establishment and validation of new complementing cells for production of E1-deletedadenovirus vectors in serum -free suspension culture. J. Virol. Methods.208:177-188.
Gossen, M., and H. Bujard. 1992. Tightcontrol of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89:5547-5551.
Grieger, JC, and RJ Samulski. 2012.Adeno-associated virus vectorology, manufacturing, and clinical applications.Methods Enzymol. 507:229-254.
Kafri, T., PH van, L. Ouyang, FH Gage,and IM Verma. 1999. A packaging cell line for lentivirus vectors. J. Virol.73:576-584.
Kotterman, MA, and DV Schaffer. 2014.Engineering adeno-associated viruses for clinical gene therapy. Nat. Rev. Genet. 15:445-451.
Massie, B., F. Couture, L. Lamoureux, DDMosser, C. Guilbault, P. Jolicoeur, F. Belanger, and Y. Langelier. 1998a. Inducible overexpression of a toxic protein by an adenovirusvector with a tetracycline-regulatable expression cassette J. Virol. 72:2289-2296.
Massie, B., DD Mosser, M. Koutroumanis, I. Vitte-Mony, L. Lamoureux, F. Couture, L. Paquet, C. Guilbault, J. Dionne, D. Chahla, P. Jolicoeur, and Y. Langelier. . 1998b. Newadenovirus vectors for protein production and gene transfer. Cytotechnology.28:53-64.
Matrai, J., MK Chuah, and T. VandenDriessche. 2010. Recent advances in lentiviral vector development and applications. Mol. Ther. 18:477-490.
Mercille, S., P. Jolicoeur, C. Gervais, D.Paquette, DD Mosser, and B. Massie. 1999.Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myelomacells transfected with the E1B-19K adenoviral gene. Biotechnol. Bioeng.63:516-528.
Milone, MC, and U. O'Doherty. 2018.Clinical use of lentiviral vectors. Leukemia. 32:1529-1541.
Miyazaki, J., S. Takaki, K. Araki, F. Tashiro, A. Tominaga, K. Takatsu, and K. Yamamura. 1989. Expression vectorsystem based on the chicken beta-actin promoter directs efficient production of interleukin-5. Gene . 79:269-277.
Miyoshi, H., U. Blomer, M. Takahashi, FHGage, and IM Verma. 1998. Development of a self-inactivating lentivirus vector. J. Virol. 72:8150-8157.
Mullick, A., Y. Xu, R. Warren, M. Koutroumanis, C. Guilbault, S. Broussau, F. Malenfant, L. Bourget, L. Lamoureux, R. Lo, AW Caron, A. Pilotte, and B Massie. 2006. The cumategene-switch: a system for regulated expression in mammalian cells. BMCBiotechnol. 6:43.
Ni, Y., S. Sun, I. Oparaocha, L. Humeau, B.Davis, R. Cohen, G. Binder, YN Chang, V. Slepushkin, and B. Dropulic. 2005.Generation of a packaging cell line for Prolonged large-scale production of high-titer HIV-1-based lentiviral vector. J. Gene Med. 7:818-834.
Pacchia, AL, ME Adelson, M. Kaul, Y.Ron, and JP Dougherty. 2001. An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins. Virology. 282:77-86.
Pluta, K., and MM Kacprzak. 2009. Use ofHIV as a gene transfer vector. Acta Biochim. Pol.56:531-595.
Poulain, A., S. Perret, F. Malenfant, A.Mullick, B. Massie, and Y. Durocher. 2017. Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch. J Biotechnol. 255:16 -27.
Robert, MA, PS Chahal, A. Audy, A.Kamen, R. Gilbert, and B. Gaillet. 2017. Manufacturing of recombinantadeno-associated viruses using mammalian expression platforms. Biotechnol. J.12.
Sanber, KS, SB Knight, SL Stephen, R.Bailey, D. Escors, J. Minshull, G. Santilli, AJ Thrasher, MK Collins, andY. Takeuchi. 2015. Construction of stable packaging cell lines for clinicallentiviral vector production. Sci Rep. 5:9021.
Sparacio, S., T. Pfeiffer, H. Schaal, andV. Bosch. 2001. Generation of a flexible cell line with regulatable, high-level expression of HIV Gag/Pol particles capable of packaging HIV-derived vectors.Mol. Ther. 3 :602-612.
Umana, P., CA Gerdes, D. Stone, JRDavis, D. Ward, MG Castro, and PR Lowenstein. 2001. Efficient FLPerecombinase enables scalable production of helper- dependent adenoviral vectorswith negligible helper-virus contamination. Nat.Biotechnol. 19: 582-585.
Vigna, E., S. Cavalieri, L. Ailles, M.Geuna, R. Loew, H. Bujard, and L. Naldini. 2002. Robustand regulation of efficient transgene expression in vivo by improvedtetracycline-dependent lentiviral vectors. Mol. Ther 5:252-261.
Weitzman, MD, and RM Linden. 2011. Adeno-associated virus biology. Methods Mol. Biol. 807:1-23.
Wright, JF 2009. Transient transfectionmethods for clinical adeno-associated viral vector production. Hum. Gene Ther.20:698-706.
Zhao, HF, J. Boyd, N. Jolicoeur, and SHShen. 2003. A coumermycin/novobiocin-regulated gene expression system. Hum.Gene Ther. 14:1619-1629.

[Cross reference with related applications]
[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/134,816, filed January 7, 2021, the contents of which are hereby incorporated by reference in their entirety. be incorporated into.

[Incorporation of sequence listing]
[0002] The computer readable form of the sequence list "P61102PC00 Sequence Listing_ST25" (26,894 bytes) created on January 5, 2022 is incorporated herein by reference.

Claims (62)

a) a first expression cassette comprising a nucleic acid molecule encoding a cumate repressor protein operably linked to a constitutive promoter and a polyadenylation signal;
b) a second expression cassette comprising a nucleic acid molecule encoding a coumermycin chimeric transactivator protein operably linked to a coumate-inducible promoter and a polyadenylation signal; and c)
i) comprising a coumermycin-inducible promoter, a cloning site, and a polyadenylation signal, the cloning site encoding a first RNA or protein of interest operably linked to the coumermycin-inducible promoter and the polyadenylation signal; or ii) a third nucleic acid molecule encoding a first RNA or protein of interest, operably linked to a coumermycin-inducible promoter and a polyadenylation signal. An inducible expression system containing an expression cassette for. The constitutive promoter is human ubiquitin C (UBC) promoter, human elongation factor 1α (EF1A) promoter, human phosphoglycerate kinase 1 (PGK) promoter, simian virus 40 early promoter (SV40), β-actin promoter, cytomegalovirus, etc. 2. The expression system of claim 1, selected from the group consisting of viral immediate early promoter (CMV), hybrid CMV enhancer/β-actin promoter (CAG), and variants thereof. 2. The cumate repressor protein comprises the amino acid sequence set forth in SEQ ID NO: 2 or a functional variant thereof, or is encoded by a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a functional variant thereof. 1 or the expression system according to claim 2. The expression system according to any one of claims 1 to 3, wherein the cumate-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO: 5 or a functional variant thereof. The coumermycin chimeric transactivator protein comprises the amino acid sequence set forth in SEQ ID NO: 14 or a functional variant thereof, or is encoded by a nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO: 13 or a functional variant thereof. , the expression system according to any one of claims 1 to 4. Any one of claims 1 to 5, wherein the coumermycin-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO: 9 or a functional variant thereof, or comprises the nucleotide sequence set forth in SEQ ID NO: 10 or a functional variant thereof. The expression system described in section. The expression system according to any one of claims 1 to 6, wherein the coumermycin-inducible promoter further comprises a tripartite leader (TPL) and/or a major late promoter (MLP) enhancer. The expression system according to claim 7, wherein the coumermycin-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO: 11 or a functional variant thereof. The expression system according to any one of claims 1 to 8, wherein the coumermycin-inducible promoter further comprises a human β-globin intron. The expression system according to claim 9, wherein the coumermycin-inducible promoter comprises the nucleotide sequence set forth in SEQ ID NO: 12 or a functional variant thereof. Any one of claims 1 to 10, wherein the third expression cassette comprises a nucleic acid molecule encoding a first RNA or protein of interest operably linked to a coumermycin inducible promoter and a polyadenylation signal. Expression system described in. 12. The expression system of claim 11, wherein the third expression cassette encodes a recombinant protein. 13. According to any one of claims 1 to 12, further comprising a fourth expression cassette comprising a nucleic acid molecule encoding a second RNA or protein of interest operably linked to a promoter and a polyadenylation signal. expression system. 14. The expression system of claim 13, further comprising a fifth expression cassette comprising a nucleic acid molecule encoding a third RNA or protein of interest operably linked to a promoter and a polyadenylation signal. The expression system according to claim 13 or 14, wherein the promoter of the fourth and/or fifth expression cassette is a coumermycin-inducible promoter. 16. The expression system according to claim 15, wherein the promoter of the fourth and/or fifth expression cassette is a constitutive promoter. Expression system according to any one of claims 11 to 14 encoding one or more components of a viral vector. the third expression cassette encodes a lentiviral REV protein, the promoter of the fourth expression cassette is a coumermycin inducible promoter, the fourth expression cassette encodes a viral envelope protein, and the fifth expression cassette encodes a viral envelope protein; Expression system according to any one of claims 14 to 17, wherein the cassette encodes a lentiviral Gag/pol. The third expression cassette encodes a viral envelope protein, the promoter of the fourth expression cassette is a coumermycin inducible promoter, the fourth expression cassette encodes a lentiviral Gag/pol, and the fifth expression cassette encodes a lentiviral Gag/pol. Expression system according to any one of claims 14 to 17, wherein the expression cassette encodes a lentiviral REV protein. Expression system according to claim 18 or 19, wherein the viral envelope protein is VSVg, optionally VSVg-Q96H-I57L. Claims 13-17, wherein the third expression cassette encodes Rep40 or Rep52 and the fourth expression cassette encodes Rep68 or Rep78, and wherein the fourth expression cassette is under the control of a coumermycin inducible promoter. The expression system according to any one of . The third expression cassette encodes Rep52, the fourth expression cassette encodes Rep68, the fifth expression cassette encodes Rep78, and the fourth and fifth expression cassettes encode a coumermycin inducible promoter. The expression system according to any one of claims 14 to 17, which is under the control of. Expression according to any one of claims 13 to 16, wherein the third expression cassette encodes an antibody heavy chain or a part thereof and the fourth expression cassette encodes an antibody light chain or a part thereof. system. A method for producing a mammalian cell for producing a target RNA or protein, the method comprising:
a) introducing into a mammalian cell an expression system and a selectable marker according to any one of claims 11 to 23; and b) selecting the cell to select cells carrying said selectable marker. A method comprising the step of selecting cells carrying the expression system by applying pressure to produce mammalian cells for producing the desired viral vector, RNA or protein. 25. The method of claim 24, further comprising: c) isolating individual cells containing the expression system; and d) culturing the individual cells to create a cell population containing the expression system. . A method for producing mammalian cells for producing RNA or protein of interest, comprising:
a) introducing into a mammalian cell a first expression cassette of an expression system according to any one of claims 1 to 23 and a first selectable marker;
b) selecting cells carrying said first expression cassette by applying selective pressure on the cells to select cells carrying the first selectable marker;
c) isolating a first individual cell containing said first expression cassette;
d) culturing said first individual cells to obtain a first cell population comprising said first expression cassette;
e) introducing into cells of said first cell population a second expression cassette of an expression system according to any one of claims 1 to 23 and a second selectable marker;
f) selecting cells carrying the second expression cassette by applying selective pressure on the cells to select cells carrying the second selectable marker;
g) isolating a second individual cell containing said second expression cassette;
h) culturing said second individual cells to obtain a second cell population comprising said second expression cassette;
i) introducing into cells of said second cell population a third expression cassette of an expression system according to any one of claims 11 to 23 and a third selectable marker;
j) selecting cells carrying said third expression cassette by applying selective pressure on the cells to select cells carrying a third selectable marker;
k) isolating a third individual cell containing said third expression cassette;
l) producing a mammalian cell for producing an RNA or protein of interest by culturing said third individual cells to obtain a third cell population comprising said third expression cassette; Including, methods. The fourth expression cassette of the expression system according to any one of claims 13 to 23, and optionally the fifth expression cassette of the expression system according to any one of claims 14 to 23, comprises step 27. The method of claim 26, wherein the method is introduced into the cell in i) or after step l). A method for producing mammalian cells for producing RNA or protein of interest, comprising:
a) A first expression cassette of an expression system according to any one of claims 1 to 23, a second expression cassette of an expression system according to any one of claims 1 to 23 to a mammalian cell. , and introducing a first selectable marker;
b) applying selective pressure to cells to select cells carrying said first selectable marker;
c) isolating a first individual cell containing said first expression cassette and second expression cassette;
d) culturing said first individual cells to obtain a first population of cells comprising said first expression cassette and said second expression cassette;
e) introducing into cells of said first cell population a third expression cassette of an expression system according to any one of claims 11 to 23 and a second selectable marker;
f) applying selective pressure to cells to select cells carrying said second selectable marker;
g) isolating a second individual cell containing said third expression cassette;
h) producing a mammal for the production of an RNA or protein of interest by culturing said second individual cells to obtain a second population of cells comprising said third expression cassette. ,Method. The fourth expression cassette of the expression system according to any one of claims 13 to 23, and optionally the fifth expression cassette of the expression system according to any one of claims 14 to 23, comprises step 29. The method of claim 28, wherein the method is introduced into the cell in e) or after step h). 24-24, wherein the expression system or one or more expression cassettes of the expression system is introduced into the cell by transfection, transduction, infection, electroporation, sonoporation, nucleofection, or microinjection. 29. The method according to any one of 29. A cell produced by the method according to any one of claims 24 to 30. A cell comprising the expression system according to any one of claims 11 to 23. 33. A cell according to claim 31 or 32, which is a human cell, optionally a DE human embryonic kidney (HEK)-293 cell or a derivative thereof. 33. The cell according to claim 31 or 32, which is a Chinese hamster ovary (CHO) cell or a derivative thereof, a VERO cell or a derivative thereof, a HeLa cell or a derivative thereof, an A549 cell or a derivative thereof, a stem cell or a derivative thereof, or a neuron or a derivative thereof. cell. 35. A method for producing RNA or protein of interest, comprising the step of culturing a cell according to any one of claims 31 to 34 in the presence of a cumate effector molecule and a coumermycin effector molecule, A method, wherein the third expression cassette of the cellular expression system according to any one of paragraphs 31 to 34 encodes an RNA or protein of interest, and the RNA or protein of interest is produced. 36. The method of claim 35, wherein the cumate effector molecule is a cumate, and optionally, the cumate is present at a concentration of about 1 to about 200 μg/ml, about 50 to about 150 μg/ml, or about 100 μg/ml. . 37. The method of claim 35 or 36, wherein the coumermycin effector molecule is coumermycin, and optionally, coumermycin is present at a concentration of about 1 to about 30 nM, about 5 to about 20 nM, or about 10 nM. 38. A method according to any one of claims 35 to 37, wherein the cells are grown in suspension and/or in the absence of serum. A virus packaging cell comprising an expression system according to any one of claims 17 to 22. A virus packaging cell according to claim 39, which is a lentivirus packaging cell comprising an expression system according to any one of claims 18 to 20. 40. A virus packaging cell according to claim 39, which is an adeno-associated virus (AAV) packaging cell comprising an expression system according to claim 21 or 22. Viral packaging cell according to any one of claims 39 to 41, further comprising a viral construct having a gene of interest. A method for producing a viral vector, the method comprising:
a) introducing a virus construct carrying a gene of interest into the virus packaging cell according to any one of claims 39 to 41 or obtaining a virus packaging cell according to claim 42; and b) producing a viral vector by culturing said cell in the presence of a cumate effector molecule and a coumermycin effector molecule. 44. The method of claim 43, wherein the cumate effector molecule is cumate and/or the coumermycin effector molecule is coumermycin. 45. The method of claim 43 or 44, wherein the virus packaging cells are grown in suspension and/or in the absence of serum. A selectable marker is introduced into cells containing the viral construct, said method comprising applying selective pressure to select cells carrying said selectable marker, and optionally selecting individual cells containing said viral construct. 46. The method of any one of claims 43-45, further comprising isolating and culturing the individual cells containing the viral construct to obtain a population of cells containing the viral construct. 47. The method according to any one of claims 43 to 46, wherein the cell is a lentiviral packaging cell according to claim 40 and the viral construct is a lentiviral construct. 47. The method of any one of claims 43-46, wherein the cell is an AAV packaging cell according to claim 41 and the viral construct is an AAV construct. 1. A method of producing a mammalian cell line capable of expression, comprising:
a) A first expression cassette of an expression system according to any one of claims 1 to 23, a second expression cassette of an expression system according to any one of claims 1 to 23 to a mammalian cell. , and introducing a first selectable marker;
b) selecting cells carrying said first and second expression cassettes of said expression system by applying selective pressure on cells to select cells carrying a first selectable marker;
c) isolating individual cells; and d) producing an expressible mammalian cell line by culturing said individual cells to produce a cell line. 1. A method of producing a mammalian cell line capable of expression, comprising:
a) introducing into a mammalian cell a first expression cassette of an expression system according to any one of claims 1 to 23 and a first selectable marker;
b) selecting cells carrying said first expression cassette by applying selective pressure on cells to select cells carrying said first selectable marker;
c) isolating a first individual cell containing said first expression cassette;
d) culturing said first cells to obtain a first cell population comprising said first expression cassette;
e) introducing into cells of said first cell population a second expression cassette of an expression system according to any one of claims 1 to 23 and a second selectable marker;
f) selecting cells carrying the second expression cassette by applying selective pressure on the cells to select cells carrying said second selectable marker;
g) isolating a second individual cell comprising said second expression cassette; and h) culturing said second individual cell and forming a second population of cells comprising said second expression cassette. 1. A method comprising the step of generating an expression capable mammalian cell line by obtaining. A mammalian cell comprising a first expression cassette of an expression system according to any one of claims 1 to 23 and a second expression cassette of an expression system according to any one of claims 1 to 23. 52. A mammalian cell according to claim 51, which is a human cell, optionally a human embryonic kidney (HEK)-293 cell or a derivative thereof. A method for producing RNA or protein of interest, comprising:
a) in a cell comprising a first expression cassette of an expression system according to any one of claims 1 to 23 and a second expression cassette of an expression system according to any one of claims 1 to 23; , introducing the third expression cassette of the expression system according to any one of claims 11 to 23 and a selectable marker;
b) selecting cells carrying the first, second and third expression cassettes of said expression system by applying selective pressure on cells to select cells carrying said selectable marker; and c ) optionally isolating individual cells comprising the first, second and third expression cassettes; culturing said individual cells to form a population of cells comprising the first, second and third expression cassettes; Step of generating;
d) culturing cells containing the first, second and third expression cassettes in the presence of a cumate effector molecule and a coumermycin effector molecule, wherein the RNA or protein of interest is produced. ,Method. 54. The method of claim 53, wherein the cumate effector molecule is cumate and/or the coumermycin effector molecule is coumermycin. 55. A method according to claim 53 or 54, wherein the cells are grown in suspension and/or in the absence of serum. a) a first plasmid comprising a first expression cassette of an expression system according to any one of claims 1 to 23;
b) a second plasmid comprising a second expression cassette of an expression system according to any one of claims 1 to 23; and c) a second plasmid comprising a second expression cassette of an expression system according to any one of claims 1 to 23; A kit comprising a third plasmid containing three expression cassettes. A kit comprising a cell according to claim 51 or 52 and a plasmid comprising the third expression cassette of the expression system according to any one of claims 1 to 23. The third expression cassette comprises a coumermycin-inducible promoter, a cloning site, and a polyadenylation signal, and the cloning site is operably linked to the coumermycin-inducible promoter and polyadenylation signal, or 58. The kit according to claim 56 or 57, which is for inserting a nucleic acid molecule encoding a protein. 58. The kit of claim 56 or 57, wherein the third expression cassette comprises a nucleic acid molecule encoding the first RNA or protein of interest operably linked to a coumermycin inducible promoter and a polyadenylation signal. A kit comprising a cell according to any one of claims 39 to 41 and a virus construct. Kit according to any one of claims 56 to 60, further comprising said cumate effector molecule, optionally a cumate, and/or a coumermycin effector molecule, optionally a coumermycin. A kit comprising a cell according to any one of claims 31-34 and 39-42, and a cumate effector molecule, optionally cumate, and/or a coumermycin effector molecule, optionally coumermycin.

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